Artificial antigen presenting cells and uses therefor

ABSTRACT

The invention relates to novel artificial antigen presenting cells (aAPCs). The aAPC comprises at least one stimulatory ligand and at least one co-stimulatory ligand where the ligands each specifically bind with a cognate molecule on a T cell of interest, thereby mediating expansion of the T cell. The aAPC of the invention can further comprise additional molecules useful for expanding a T cell of interest. The aAPC of the invention can be used as an “off the shelf” APC that can be readily designed to expand a T cell of interest. Also, the aAPC of the invention can be used identify the stimulatory, co-stimulatory, and any other factors that mediate growth and expansion of a T cell of interest. Thus, the present invention provides powerful tools for development of novel therapeutics where activation and expansion of a T cell can provide a benefit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/137,807, now U.S. Pat. No. 7,754,482 filed May 25, 2005, which claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationNo. 60/575,712, filed May 27, 2004, all of which are hereby incorporatedby reference in their entirety herein.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH OR DEVELOPMENT

This invention was made with government support under National Instituteof Health Grants R21 AI060477, R01 CA105216 and R01 AI057858. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adoptive transfer is a term coined by Medawar (1954, Proc. Royal Soc.143:58-80) to study allograft rejection. The term adoptive immunotherapydenotes the transfer of immunocompetent cells for the treatment ofcancer or infectious diseases (June, C. H., ed., 2001, In: CancerChemotherapy and Biotherapy: Principles and Practice, LippincottWilliams & Wilkins, Baltimore; Vonderheide et al., 2003, Immun. Research27:1-15). Adoptive therapy can be considered as a strategy aimed atreplacing, repairing, or enhancing the biological function of a damagedtissue or system by means of autologous or allogeneic cells. The firstsuccessful infusion of ex vivo expanded, HIV infected, polyclonal CD4 Tcells that enabled a high degree of engraftment upon infusion, wasperformed using magnetic beads coated with anti-CD3 and anti-CD28 beads(CD3/28 coated beads) to ex vivo expand the HIV infected individuals Tcells. Eight patients were administered 51 infusions of costimulated CD4cells under this protocol with minimal adverse advents (Levine et al.,2002, Nature Med. 8:47-53).

HIV infection induces a pronounced expansion of HIV-specific CD8 Tcells. These CD8 T cells release soluble factors (Walker et al., 1986,Science 234:1563-1566; Zhang et al., 2002, Science 298:995-1000; Cocchiet al., 1995, Science 270:1811-1815) that limit HIV replication as wellas directly lyse HIV infected cells (Walker et al., 1987, Nature328:345-348; Koup et al., 1994, J. Virol. 68:4650-4655). Depletion ofCD8 T cells prior to SIV challenge leads to unchecked viral replicationand a rapid death, indicating that CD8 T cell activity is necessary tomake HIV a chronic disease (Schmitz et al. 1999, Science 283:857-860;Jin et al., 1999, J. Exp. Med. 189:991-998). Yet, CD8 T cells ultimatelyfail to control HIV infection. HIV-specific T cells have been shown tohave highly reduced perforin expression (Zhang et al., 2003, Blood101:226-235; Appay, et al., 2000, J. Exp. Med. 192:63-75),down-regulation of two key signaling receptors, CD3ζ and CD28 (Trimbleet al., 2000, Blood 96:1021-1029), a skewed maturation pattern (Appay etal., 2002, Nature Med. 8, 379-385) and a high sensitivity to Fas inducedapoptosis (Mueller et al., 2001, Immunity, 15:871-882). Thus, it isbelieved that optimal activation of HIV specific CD8 T cells willrestore effector functions.

Anti-CD3 and anti-CD28 (CD3/28) coated beads were the first generationof artificial APCs (aAPC) that permitted expansion of HIV-infected CD4 Tcells (Levine et al., 1996, Science 272:1939-1943). In addition todelivering the signals needed for T cell activation and growth, CD3/28bead stimulation renders T cells resistant to R5 infection bydown-regulating CCR5 and up-regulating the expression of its ligands,the β-chemokines RANTES, Macrophage Inflammatory Protein-1 alpha(MIP-1α) and MIP-1β (Riley et al., 1997, J. Immunol. 158:5545-5553Carroll et al., 1997, Science 276:273-276). Several phase I and IItrials have demonstrated that infusion of autologous CD4 T cellsexpanded using CD3/28 coated beads into R5-infected individuals is bothsafe and feasible (Carroll et al., 1997, Science 276:273-276; Levine etal., 2002, Nature Med. 8:47-53; Walker et al., 2000, Blood 96:467-474;Ranga et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:1201-1206). Moreimportantly, sustained increases in the total lymphocyte count, the CD4to CD8 T cell ratio, the fraction of cytokine-secreting T cells, and theability to respond to recall antigens were observed, suggesting thatadoptive T cell immunotherapy can restore at least limited immunefunction back to HIV infected individuals (Levine et al., 2002, NatureMed. 8:47-53). Despite the success of these initial trials, severallimitations were noted, including the difficulty of (1) expanding CD8 Tcells, (2) adding additional costimulatory signals that may be requiredto expand certain subsets of T cells, (3) removing the beads beforeinfusion and (4) generating antigen specific T cells with a highengraftment potential.

Others have used T cell expansion CD3/28 coated beads to introduce genemodified T cells to HIV infected patients. In these studies (Walker etal., 2000, Blood 96:467-474; Mitsuyasu et al., 2000, Blood 96:785-793;Deeks et al., 2002, Mol. Ther. 5:788-797), a chimeric moleculeconsisting of the extracellular domain of CD4 and the intracellulardomain of the CD3 zeta chain (CD4ζ was introduced into CD4 T cells viaretroviral transduction). CD4ζ-modified T cells were detected by DNA PCRin the peripheral blood of all patients following infusion, and meanlevels of 1-3% of total peripheral blood mononuclear cells (PBMCs) weresustained at all post-infusion time points. In an extended follow-up,CD4ζ was detected in the blood of 17 of 18 patients one year followinginfusion. These levels of sustained engraftment are several orders ofmagnitude higher than what has been previously observed for human T cellinfusions, perhaps because previous cell culture techniques may haveinduced susceptibility to apoptosis or replicative senescence (Rosenberget al., 1990, N. Engl. J. Med. 323:570-578; Yee et al., 2002, Proc.Natl. Acad. Sci. U.S.A. 99:16168-16173; Brodie et al., 1999, Nature Med.5:34-41; Riddell et al., 1996, Nature Med. 2:216-223; Riddell et al.,2000, Cancer Journal 6:S250-S258). These clinical results indicate thatcostimulated T cells propagated with bead-based aAPCs are safe and havethe capacity for prolonged engraftment. However, due to the limitednumber of study subjects and length of time required to achieve aclinical endpoint in a HIV therapeutic trial, statistical significanceof the clinical benefit of autologous CD4 T cell transfer to HIVinfected individuals could not be measured.

While potentially effective in limiting immunodeficiency, polyclonal CD4T cells are likely to have only a modest effect on the HIV specificresponse. Immunotherapy of human viral infection using adoptive transferof antigen-specific CD8 T cells has been studied in the setting of CMV,EBV, and HIV infection. This approach has been evaluated using T cellclones with HLA-restricted antigenic specificity for CMV (Riddell etal., 1992, Science 257:238-241; Walter et al., 1995, N. Engl. J. Med.333:1038-1044). CMV-specific CD8⁺ T cells isolated from MHC-identicalbone marrow donors were expanded ex vivo and were administered to 14allogeneic bone marrow transplant recipients. Recovery of CMV-specificCTL activity was seen in each case and adoptively transferred CTLpersisted in vivo for up to 12 weeks. In a similar study, donor-derivedEBV-specific CD8⁺ and CD4⁺ T cells, genetically marked with the neomycinresistance gene, were administered to 6 recipients of T cell-depletedallogeneic bone marrow allografts (Rooney et al., 1995, Lancet 345:9-13;Heslop et al., 1996, Nature Med. 2:551-555). Gene-marked CD4⁺ and CD8⁺ Tcells responsive to in vivo or ex vivo challenge with EBV persisted atlow frequencies in vivo for as long as 18 months after infusion.Infusion of CD8 T cells with a single specificity to HIV (Nef) (Koeniget al., 1995, Nature Med. 1:330-336) into one patient demonstrated CTLselection of viral variants indicating that infusion of HIV specific Tcells against multiple targets may be necessary to control HIVreplication. In all of these studies, the inability of the vast majorityof these T cells to engraft has limited the study of the long-termeffects of antigen specific CD8 T cell immunotherapy. A major challengein the field is to expand CD8 T cells that will engraft and have potenteffector functions on a long-term basis to more effectively fightchronic infections. However, despite the long-term need for sufficientnumbers of therapeutic T cells, there are no methods available forexpanding these cells.

HIV specific T cells are able to contain but not eradicate HIV. Studiesthat have removed CD8 T cells prior to, or during, HIV infection havedemonstrated unchecked viral replication and a much faster progressionto AIDS, indicating that CD8 T cells are important in controlling HIV(Schmitz et al. 1999, Science 283:857-860; Jin et al., 1999, J. Exp.Med. 189:991-998). However, HIV specific T cells in general, lackperforin expression (Gandhi et al., 2002, Annu. Rev. Med. 53:149-172)and other requisite effector functions to eliminate HIV from the host.Studies of long term non-progressors indicated that HIV specific T cellsfrom these individuals are more likely to proliferate and containperforin, demonstrating that CD8 T cells with enhanced effectorfunctions may delay the progression to AIDS (Migueles et al., 2002,Nature Immunol. 3:1061-1068). Other investigators have demonstrated thattwo key signaling receptors, CD3ζ and CD28 are downregulated on HIVspecific T cells (Trimble et al., 2000, Blood 96:1021-1029; Trimble etal., 1998, Blood 91:585-594; Trimble et al., 2000, J. Virol.74:7320-7330), and that HIV-specific T cells are more sensitive to Fasinduced apoptosis (Mueller et al., 2001, Immunity, 15:871-882). Appay etal. (2002, Nature Med. 8:379-385) examined the differences between HIV-,EBV-, and CMV-specific CD8 T cells based on CD27 and CD28 expression.Early differentiated T cells expressed both CD27 and CD28 and possessedpoor effector functions but excellent proliferative abilities.Intermediate T cells were CD27 positive but CD28 negative, and thesecells had limited proliferative and effector functions. The mostdifferentiated T cells lack both CD27 and CD28, and these cells hadlittle proliferative ability but enhanced effector functions. It wasobserved that most of the HIV-specific T cells had the intermediatephenotype. Therefore, the cells being “stuck” in this intermediate Tcell phenotype that lack both proliferative capacity and effectorfunctions may be the contributing factor to the ineffectiveness ofHIV-specific T cells (Appay et al., 2002, Nature Med. 8, 379-385).Moreover, CD8 T cell function is highly dependent upon CD4 T cellfunction (Zajac et al., 1998, J. Exp. Med. 188:2205-2213; Shedlock etal., 2003, Science 300:337-339; Sun et al., 2003, Science 300:339-342)and since HIV targets CD4 T cells, the CD8 T cell defects observed inHIV infection could be the result of a lack of T cell help.

Thus, there exists a long-felt need to provide ways to stimulate T cellsto combat various acute and chronic diseases and to promulgatesufficient numbers of therapeutic T cells for adoptive immunotherapy.The present invention meets this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention includes an isolated artificial antigen presentingcell (aAPC), said aAPC comprising a K562 cell transduced using alentiviral vector (LV), wherein said LV comprises a nucleic acidencoding at least one immune stimulatory ligand and at least oneco-stimulatory ligand and further wherein said aAPC expresses saidstimulatory ligand and said co-stimulatory ligand and can stimulate andexpand a T cell contacted with said aAPC.

In one aspect of the present invention, the stimulatory ligand is apolypeptide selected from the group consisting of a majorhistocompatibility complex Class I (MHC class I) molecule loaded with anantigen, an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2antibody.

In another aspect of the present invention, said co-stimulatory ligandis at least one co-stimulatory ligand selected from the group consistingof CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L,ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, ILT3, ILT4, 3/TR6, and a ligand that specifically binds withB7-H3.

In yet another aspect of the present invention, said co-stimulatoryligand specifically binds with at least one of a co-stimulatory moleculeselected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, BTLA, Tollligand receptor and a ligand that specifically binds with CD83.

In still another aspect of the present invention, said co-stimulatoryligand is an antibody that specifically binds with at least one moleculeselected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligandreceptor and a ligand that specifically binds with CD83.

In one aspect of the present invention, said aAPC further comprises Fcγreceptor selected from the group consisting of a CD32 molecule and aCD64 molecule.

In another aspect of the present invention, said LV comprises a nucleicacid encoding at least one antigen selected from the group consisting ofa tumor antigen, a viral antigen, a bacterial antigen, a peptide-MHCtetramer, a peptide-MHC trimer, a peptide-MHC dimer, and a peptide-MHCmonomer.

In yet another aspect of the present invention, said tumor antigen isselected from the group consisting of MAGE-1, MAGE-2, MAGE-3, MART-1,GP100, CEA, HER-2/Neu, PSA, WT-1, MUC-1, MUC-2, MUC-3, MUC-4, andtelomerase.

In still another aspect of the present invention, said LV comprises anucleic acid encoding at least one peptide selected from a cytokine anda chemokine.

In yet another aspect of the present invention, said cytokine is atleast one cytokine selected from the group consisting of IL-2, IL-4,IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon-alpha (IFNα),interferon-beta (IFNβ), interferon-gamma (IFNγ), tumor necrosisfactor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocytemacrophage colony stimulating factor (GM-CSF), and granulocyte colonystimulating factor (GCSF).

The present invention includes a method for specifically inducingproliferation of a T cell expressing a known co-stimulatory molecule,said method comprising contacting said T cell with an aAPC of theinvention, further wherein said co-stimulatory ligand specifically bindswith said known co-stimulatory molecule, thereby specifically inducingproliferation of said T cell.

The present invention includes a method for specifically inducingproliferation of a T cell expressing a known co-stimulatory molecule,said method comprising contacting a population of T cells comprising atleast one T cell expressing said known co-stimulatory molecule with anaAPC of the invention, wherein said aAPC expresses at least oneco-stimulatory ligand that specifically binds with said knownco-stimulatory molecule, wherein binding of said known co-stimulatorymolecule with said co-stimulatory ligand induces proliferation of said Tcell.

The present invention includes a method of specifically expanding a Tcell population subset, said method comprising contacting a populationof T cells comprising at least one T cell of said subset with an aAPC ofthe invention, wherein said aAPC comprises at least one co-stimulatoryligand that specifically binds with a co-stimulatory molecule on said Tcell of said subset, wherein binding of said co-stimulatory moleculewith said co-stimulatory ligand induces proliferation of said T cell ofsaid subset, thereby specifically expanding a T cell population subset.

The present invention includes a method of identifying a co-stimulatoryligand, or combination thereof, that specifically induces activation ofa T cell subset, said method comprising contacting a population of Tcells with an aAPC of the invention, and comparing the level ofproliferation of said T cell population with the level of proliferationof an otherwise identical population of T cells not contacted with saidaAPC, wherein a greater level of proliferation of said T cells contactedwith said aAPC compared with the level of proliferation of saidotherwise identical population of T cells not contacted with said aAPC,is an indication that said co-stimulatory ligand specifically inducesactivation of said T cell.

The present invention includes a method for inducing a T cell responseto an antigen in a mammal, said method comprising administering to saidmammal the aAPC of the invention, wherein said aAPC further comprises anMHC Class I molecule loaded with said antigen, wherein said aAPC inducesproliferation of a T cell specific for said antigen, thereby inducing aT cell response to said antigen in said mammal.

The present invention includes a method of inducing a T cell response toan antigen in a mammal in need thereof, said method comprising obtaininga population of cells from said mammal wherein said population comprisesT cells, contacting said population of cells with an aAPC of theinvention, wherein said aAPC further comprises an MHC Class I complexloaded with said antigen, whereby contacting said cells with said aAPCinduces proliferation of an antigen-specific T cell specific for saidantigen, isolating said antigen-specific T cell from said population ofcells, and administering said antigen-specific T cells to said mammal,thereby inducing a T cell response to said antigen in said mammal.

The present invention includes a method of specifically expanding apopulation of T regulatory (Treg) cells, the method comprisingcontacting said population with an aAPC of claim 1, wherein said aAPCfurther comprises an Fcγ receptor loaded with an anti-CD3 antibody andan anti-CD28 antibody, the method further comprising contacting saidpopulation of cells with a cytokine, wherein binding of said anti-CD3antibody and said anti-CD28 antibody with said Treg cells inducesproliferation of said Treg cells, thereby specifically expanding apopulation of Treg cells.

In one aspect of the invention, said cytokine is interleukin-2.

The present invention includes a kit for specifically inducingproliferation of a T cell expressing a known co-stimulatory molecule,said kit comprising an effective amount of an aAPC, wherein said aAPCcomprises a K562 cell transduced using a lentiviral vector (LV), whereinsaid LV comprises a nucleic acid encoding at least one co-stimulatoryligand that specifically binds said known co-stimulatory molecule,wherein binding of said known co-stimulatory molecule with saidco-stimulatory ligand stimulates and expands said T cell, said kitfurther comprising an applicator and an instructional material for theuse of said kit.

The present invention includes a kit for specifically inducingproliferation of a T cell expressing a known stimulatory molecule, saidkit comprising an effective amount of an aAPC, wherein said aAPCcomprises a K562 cell transduced using a lentiviral vector (LV) whereinsaid LV comprises a nucleic acid encoding at least one stimulatoryligand that specifically binds said known stimulatory molecule, whereinbinding of said known stimulatory molecule with said stimulatory ligandstimulates and expands said T cell, said kit further comprising anapplicator and an instructional material for the use of said kit.

The present invention includes a kit for specifically expanding a T cellpopulation subset, said kit comprising an effective amount of an aAPC,wherein said aAPC comprises a K562 cell transduced using a lentiviralvector (LV), wherein said LV comprises a nucleic acid encoding at leastone co-stimulatory ligand that specifically binds a co-stimulatorymolecule on said T cell population, wherein binding of saidco-stimulatory molecule with said co-stimulatory ligand stimulates andexpands said T cell population, said kit further comprising anapplicator and an instructional material for the use of said kit.

The present invention includes a kit for identifying a co-stimulatoryligand, or combination of said ligands, that specifically inducesactivation of a T cell subset, said kit comprising a plurality of aAPCswherein each said aAPC comprises a K562 cell transduced using alentiviral vector (LV), wherein said LV comprises a nucleic acidencoding at least one known co-stimulatory ligand that specificallybinds with a co-stimulatory molecule, said kit further comprising anapplicator and an instructional material for the use of said kit.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a diagram illustrating a model for the construction of a Tcell culture system based on artificial antigen-presenting cells (aAPC)using a parental K562 human erythromyeloid cell line. FIG. 1 depicts anengineered K32/4-1BBL aAPC interacting with a CD8+ T cell.

FIG. 2, comprising FIGS. 2A through 2C, depicts the expansion oftetramer sorted influenza virus (flu) specific central memory T cellsusing K32/4-1BBL/CD3/28 aAPC. FIG. 2A depicts a series of imagesdemonstrating the sorting of CD8 T cells from an HLA-A2 donor who hadbeen exposed to the flu virus. FIG. 2B is a graph demonstrating thatthese cells were able to be maintained in culture for 70 days. The totalnumber of cells that would have accumulated if no cells have beendiscarded is depicted as a semi-log plot of total cell number versusdays in culture. FIG. 2C demonstrates a chromium release assay usingloaded TAP deficient HLA-A2 positive T2 cells with either the flupeptide or leaving them unpulsed at the indicated Effector to Targetratios.

FIG. 3, comprising FIGS. 3A through 3E, illustrates the creation of anaAPC that can be used to expand flu specific T cells. FIG. 3 depicts aseries of five (5) images demonstrating FACS analysis for each of themarkers CD32 (FIG. 3A), KA2 (FIG. 3B), 4-1BBL (FIG. 3D), and FluGFP(green fluorescence protein, FIG. 3E) by aAPCs and also depicting anisotype control.

FIG. 4 is a diagram illustrating an experimental model demonstratingmethods of expanding HIV specific CD8 T cells with a broad specificity.

FIG. 5 is a graph demonstrating that K562-based aAPCs (e.g., K32/CD3/28,K32/86/CD3) mediate long-term growth of CD4 T cells and do so moreeffectively than U937-based aAPCs (U32/CD3/28) or bead-based aAPCs(CD3/28 coated beads).

FIG. 6, comprising FIGS. 6A through 6D, is a graph demonstratinginduction of cytokine and costimulatory gene expression on K562-basedaAPCs but not U937-based aAPCs. FIG. 6A is a graph depicting that thelevel of induction of interleukin 15 (IL-15) by K32/CD3/28, K32/CD3,K32, U32/CD3/28, U32/CD3, U32, and resting CD4 T cells. FIG. 6B depictsPD-L1 induction in the cells, demonstrating that the K32/CD3/28 aAPCsexpress substantially higher levels of PD-L1. FIG. 6C is a graphdepicting induction of PD-L2 by various aAPCs as described supra. FIG.6D is a graph depicting induction of B7-H3 by various aAPCs.

FIG. 7 is a diagram depicting the growth rate of lentivirus (LV)transduced aAPCs cultured in Aim V media (Invitrogen, Carlsbad, Calif.)supplemented with 3% Human Ab serum (Valley Biomedical, Winchester,Va.). Various K562-based aAPCs were produced by transducing parentalK562 (k562cc; dark diamonds) using the following LV vectors constructs:KT32 (1 gene; dark squares); KT32/4-1BBL/CD86 (3 genes; lighttriangles); KT32/4-1BBL/CD86/A2/Flu-GFP (5 genes; “X”). The total numberof cells that would have accumulated if no cells have been discarded isdepicted as a semi-log plot of total cell number versus days in culture.

FIG. 8, comprising FIGS. 8A through 8E, is a graph depicting the stableco-expression of at least eight (8) genes in a single K562 aAPC(8-THREAT). The following genes were transduced into a K562 cell andwere stably expressed, as detected using flow cytometry: Flu-GFP (FIG.8A); CD80 (FIG. 8B); CD86 (FIG. 8C); 4-1BBL (FIG. 8D); and HLA ABC (FIG.8E).

FIG. 9 is a graph demonstrating long term expansion of polyclonal CD8 Tcells using LV-transduced aAPCs.

FIG. 10, comprising FIGS. 10A-10E, is a series of graphs demonstratingthat K32/4-1 BBL aAPC expanded hTERT specific cytotoxic lymphocytes(CTL). FIGS. 10A through 10C are graphs depicting the increasingpercentage of tet+ CD8 CTLs during expansion by K32/4-1BBL aAPC. Thetiming of the MoFlo sorting corresponding to each FIG. 10A-10C isindicated on the graph showing population doublings (FIG. 10D). FIG. 10Dis a graph depicting the expansion of hTERT-specific CTLs by the aAPC,where the CTLs were obtained from a breast cancer patient vaccinatedwith hTERT. FIG. 10E is a graph demonstrating that the hTERT specificCTLs expanded using the aAPC specifically lyse carcinoma cellsexpressing HLA-A2 and telomerase+ (OV-7) but not carcinoma cells thatare telomerase+ but that are HLA-A2-(SK-OV-3).

FIG. 11, comprising FIG. 11A through FIG. 11D, depicts data comparingtransfection of aAPCs using a plasmid with expression of moleculestransduced into an otherwise identical aAPC using an LV. FIG. 11A is adiagram depicting an exemplary LV used herein, depicting the particularmodifications as disclosed elsewhere herein. FIGS. 11B and 11C depictthe transduction efficiency of K562 cells of a single inoculum of GFPexpressing virus (monocistronic) or mCD8 IRES GFP (bicistronic). Surfaceexpression of mCD8 and GFP was measured 5 days after transduction.

FIG. 12, comprising FIGS. 12A through 12F, depicts a representativeexperiment, wherein 8,000 antigen-specific T cells were observed toyield 2X 106 cells after one month of culture. Briefly, purified T cellsobtained from an HLA A*0201 donor were stained with anti-CD8 mAb and anA*0201 MHC tetramer complexed to an A*0201 restricted epitope of theinfluenza matrix protein (flu-MP tetramer). The tetramer positivepopulation (about 8000 cells) was sorted and stimulated with irradiatedKTA2/CD32/4-1BBL/FLU GFP aAPCs that were loaded with anti-CD28 antibody.The cells were re-stimulated with KTA2/CD32/4-1BBL/FLU GFP aAPCs (FIG.12A-D) approximately every 10-12 days. Interleukin 2 (IL-2) was added tothe culture at every cell feeding (every 2-3 days). FIGS. 12E and 12Fgraphs demonstrating purity of flu tetramer reactive cells prior andafter 26 days of expansion. That is, about 250-fold expansion oftetramer positive population was observed under these cultureconditions.

FIG. 13 is a bar graph depicting the level of cytokine expression usinga K562 aAPC transduced with CD32 IRES IL-7 (KT32-IL7) and K562transduced with CD32 IRES IL-15 (KT32-IL15) where the cells were sortedfor high CD32 expression.

FIG. 14 is a plot demonstrating the stable expression of CD64 on thesurface of a K562 cell transduced with a lentiviral vector expressingCD64.

FIG. 15 is a graph illustrating the increased antibody binding capacityof K562 cells transfected with a lentiviral vector expressing CD64 (K64cells).

FIG. 16 is a series of graphs illustrating that K64 cells loaded withantibody and washed multiple times are superior at stimulating T cellswhen compared with K562 cells expressing CD32 (K32 cells), although bothK64 cells and K32 cells are capable of stimulating T cells.

FIG. 17, comprising FIGS. 17A through 17D is a series of imagesillustrating that much less antibody is required to optimally load K64cells when compared to K32 cells. FIGS. 17A and 17B depict CD4 cells,FIGS. 17C and 17D depict CD8 cells.

FIG. 18 is a graph illustrating the increased expansion of Treg cellsstimulated with K32 cells loaded with anti-CD3 and anti-CD28 antibodiescompared to Treg cells stimulated with anti-CD3 and anti-CD28 coatedbeads.

FIG. 19 is a graph depicting the ability of Treg cells to suppress anallogeneic mixed lymphocyte reaction (MLR).

FIG. 20 is a graph demonstrating the CD4⁺ CD25⁺ Treg cells stimulatedusing K32 cells expressing OX40L render the Treg cells non-suppressive.

FIG. 21, comprising FIGS. 21A through 21C, is a series of imagesillustrating the expansion of antigen specific CD8 cells using K32loaded with anti-CD3 antibody and expressing IL-15, 4-1BBL and CD80.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the surprising discovery that lentivirusvectors can be used to efficiently produce aAPCs that stably expressnumerous T cell stimulatory and co-stimulatory ligands, and antibodiesthereto, as well as antigens, cytokines, among other molecules. Theinvention also relates to the novel aAPCs produced and methods for theiruse to expand a desired T cell, activate and/or expand specific T cellsubsets, identify stimulatory molecules, co-stimulatory molecules, andcombinations thereof, that can promote expansion of specific T cellsubsets, as well as numerous therapeutic uses relating to expansion andstimulation of T cells using the novel aAPCs.

As demonstrated by the data disclosed herein, upon T cell activation,factors such as IFNγ are secreted that in turn induce the expression ofcytokines such as IL-15 and costimulatory ligands such as B7-H3 in K562cells (Thomas et al., 2002, Clin. Immunol. 105:259-272). The interchangeor “crosstalk” between the aAPC and a T cell is a reason why cell-basedaAPCs are more efficient T cell expansion systems than bead-based aAPCs.K562 cells are engineered such that these cells are a continuouslyrenewable “off the shelf” dendritic cell (DC) replacement system. Use ofaAPCs would obviate the time and expense required to generate autologousDC as a source of APC for cell culture. Additional costimulatory signalsmay be necessary to rescue effector functions from HIV-specific CD8 Tcells and as demonstrated by the data herein, aAPC cells can be modifiedto express such signals as desired. Again, this is an advantage overbead-based systems which do not encompass adding additionalcostimulatory signals that may be required to expand certain subsets ofT cells.

Previously, cell-based aAPCs were created by electroporation of K562cells with CD32 and 4-1BBL expression plasmids. Using a combination ofdrug selection, cell sorting, and limiting dilution, high-expressingclones were isolated (Maus et al., 2002, Nature Biotechnol. 20:143-148).While effective, this approach is both time consuming and limited by theneed to use drug selection markers. Reliance of drug selection restrictsthe number of constructs that can be introduced into K562 cells andraises GMP compliance issues when clinical use is contemplated.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

As used herein, “amino acids” are represented by the full name thereof,by the three-letter code corresponding thereto, or by the one-lettercode corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding a protein,or to a sequence which is substantially homologous to the non-codingstrand. As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for administering the compounds and compositions of theinvention.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated,then the animal's health continues to deteriorate. In contrast, a“disorder” in an animal is a state of health in which the animal is ableto maintain homeostasis, but in which the animal's state of health isless favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

By the term “effective amount”, as used herein, is meant an amount thatwhen administered to a mammal, causes a detectable level of T cellresponse compared to the T cell response detected in the absence of thecompound. T cell response can be readily assessed by a plethora ofart-recognized methods.

The skilled artisan would understand that the amount of the compound orcomposition administered herein varies and can be readily determinedbased on a number of factors such as the disease or condition beingtreated, the age and health and physical condition of the mammal beingtreated, the severity of the disease, the particular compound beingadministered, and the like.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in the kit for effecting alleviating ortreating the various diseases or disorders recited herein. Optionally,or alternately, the instructional material may describe one or moremethods of alleviating the diseases or disorders in a cell or a tissueor a mammal, including as disclosed elsewhere herein.

The instructional material of the kit may, for example, be affixed to acontainer that contains the compound and/or composition of the inventionor be shipped together with a container which contains the compoundand/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that therecipient uses the instructional material and the compoundcooperatively.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

By “complementary to a portion or all of the nucleic acid encoding” aprotein of the invention, is meant a sequence of nucleic acid which doesnot encode a, e.g., costimulatory ligand protein. Rather, the sequencewhich is being expressed in the cells is identical to the non-codingstrand of the nucleic acid encoding the protein and thus, does notencode the protein.

The terms “complementary” and “antisense” as used herein, are notentirely synonymous. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand.

“Complementary” as used herein refers to the broad concept of subunitsequence complementarity between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of an mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anticodon regionof a transfer RNA molecule during translation of the mRNA molecule orwhich encode a stop codon. The coding region may thus include nucleotideresidues corresponding to amino acid residues which are not present inthe mature protein encoded by the mRNA molecule (e.g., amino acidresidues in a protein export signal sequence).

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., retroviruses, lentiviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

A first region of an oligonucleotide “flanks” a second region of theoligonucleotide if the two regions are adjacent one another or if thetwo regions are separated by no more than about 1000 nucleotideresidues, and preferably no more than about 100 nucleotide residues.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 18 nucleotides in length, preferably, atleast about 24 nucleotides, more typically, from about 24 to about 50nucleotides, preferably, at least about 50 to about 100 nucleotides,even more preferably, at least about 100 nucleotides to about 200nucleotides, yet even more preferably, at least about 200 to about 300,even more preferably, at least about 300 nucleotides to about 400nucleotides, yet even more preferably, at least about 400 to about 500,and most preferably, the nucleic acid fragment will be greater thanabout 500 nucleotides in length.

As applied to a protein, a “fragment” of a stimulatory or costimulatoryligand protein or an antigen, is about 6 amino acids in length. Morepreferably, the fragment of a protein is about 8 amino acids, even morepreferably, at least about 10, yet more preferably, at least about 15,even more preferably, at least about 20, yet more preferably, at leastabout 30, even more preferably, about 40, and more preferably, at leastabout 50, more preferably, at least about 60, yet more preferably, atleast about 70, even more preferably, at least about 80, and morepreferably, at least about 100 amino acids in length amino acids inlength.

A “genomic DNA” is a DNA strand which has a nucleotide sequencehomologous with a gene as it exists in the natural host. By way ofexample, a fragment of a chromosome is a genomic DNA.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arecompletely or 100% homologous at that position. The percent homologybetween two sequences is a direct function of the number of matching orhomologous positions, e.g., if half (e.g., five positions in a polymerten subunits in length) of the positions in two compound sequences arehomologous then the two sequences are 50% identical, if 90% of thepositions, e.g., 9 of 10, are matched or homologous, the two sequencesshare 90% homology. By way of example, the DNA sequences 5′ATTGCC3′ and5′TATGGC3′ share 50% homology.

In addition, when the terms “homology” or “identity” are used herein torefer to the nucleic acids and proteins, it should be construed to beapplied to homology or identity at both the nucleic acid and the aminoacid sequence levels.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

Preferably, when the nucleic acid encoding the desired protein furthercomprises a promoter/regulatory sequence, the promoter/regulatory ispositioned at the 5′ end of the desired protein coding sequence suchthat it drives expression of the desired protein in a cell. Together,the nucleic acid encoding the desired protein and itspromoter/regulatory sequence comprise a “transgene.”

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “polyadenylation sequence” is a polynucleotide sequence which directsthe addition of a poly A tail onto a transcribed messenger RNA sequence.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

A “portion” of a polynucleotide means at least at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe” refers to a polynucleotide that is capable of specificallyhybridizing to a designated sequence of another polynucleotide. A probespecifically hybridizes to a target complementary polynucleotide, butneed not reflect the exact complementary sequence of the template. Insuch a case, specific hybridization of the probe to the target dependson the stringency of the hybridization conditions. Probes can be labeledwith, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the term “reporter gene” means a gene, the expression ofwhich can be detected using a known method. By way of example, theEscherichia coli lacZ gene may be used as a reporter gene in a mediumbecause expression of the lacZ gene can be detected using known methodsby adding a chromogenic substrate such as o-nitrophenyl-β-galactoside tothe medium (Gerhardt et al., eds., 1994, Methods for General andMolecular Bacteriology, American Society for Microbiology, Washington,D.C., p. 574).

A “restriction site” is a portion of a double-stranded nucleic acidwhich is recognized by a restriction endonuclease.

A first oligonucleotide anneals with a second oligonucleotide “with highstringency” if the two oligonucleotides anneal under conditions wherebyonly oligonucleotides which are at least about 73%, more preferably, atleast about 75%, even more preferably, at least about 80%, even morepreferably, at least about 85%, yet more preferably, at least about 90%,and most preferably, at least about 95%, complementary anneal with oneanother. The stringency of conditions used to anneal twooligonucleotides is a function of, among other factors, temperature,ionic strength of the annealing medium, the incubation period, thelength of the oligonucleotides, the G-C content of the oligonucleotides,and the expected degree of non-homology between the twooligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g., Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

As used herein, the term “transgene” means an exogenous nucleic acidsequence which exogenous nucleic acid is encoded by a transgenic cell ormammal.

A “recombinant cell” is a cell that comprises a transgene. Such a cellmay be a eukaryotic cell or a prokaryotic cell. Also, the transgeniccell encompasses, but is not limited to, an aAPC, an embryonic stem cellcomprising the transgene, a cell obtained from a chimeric mammal derivedfrom a transgenic ES cell where the cell comprises the transgene, a cellobtained from a transgenic mammal, or fetal or placental tissue thereof,and a prokaryotic cell comprising the transgene.

By the term “exogenous nucleic acid” is meant that the nucleic acid hasbeen introduced into a cell or an animal using technology which has beendeveloped for the purpose of facilitating the introduction of a nucleicacid into a cell or an animal.

By “tag” polypeptide is meant any protein which, when linked by apeptide bond to a protein of interest, may be used to localize theprotein, to purify it from a cell extract, to immobilize it for use inbinding assays, or to otherwise study its biological properties and/orfunction.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease (i.e., viral infection, tumor growth and/ormetastasis, or other effect mediated by decreased numbers and/ordecreased activity of T cells, and the like) are experienced by apatient.

By the term “vector” as used herein, is meant any plasmid or virusencoding an exogenous nucleic acid. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into virions or cells, such as, for example, polylysinecompounds and the like. The vector may be a viral vector which issuitable as a delivery vehicle for delivery of a nucleic acid thatencodes a protein and/or antibody of the invention, to the patient, orto the aAPC, or the vector may be a non-viral vector which is suitablefor the same purpose.

Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, a lentiviral vector, arecombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5:3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

A “therapeutic” treatment is a treatment administered to a patient whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs and/or decreasing or diminishing the frequency,duration and intensity of the signs.

An “effective amount” of a compound is that amount of a cell (e.g., anaAPC or T cell stimulated and/or expanded thereby) which is sufficientto provide a detectable effect to a population of T cells, or to amammal, to which the aAPC is administered and/or contacted with whencompared to an otherwise identical population of T cells, or mammal, towhich the aAPC, or T cell expanded thereby, is not administered.

The skilled artisan would understand that the effective amount variesand can be readily determined based on a number of factors such as thedisease or condition being treated, the age and health and physicalcondition of the mammal being treated, the severity of the disease, theparticular compound or cell being administered, the level of activity orexpression of the aAPC or T cell expanded thereby, and the like.Generally, the effective amount will be set between about 0.1 mg/kg toabout 100 mg/kg, more preferably from about 1 mg/kg and 25 mg/kg. Thecompound or cell (e.g., a cytokine, a stimulatory molecule or ligandthereto, a costimulatory molecule or ligand thereto, an antibody thatspecifically binds with a ligand, a nucleic acid encoding such proteins,an aAPC, a T cell expanded thereby, and the like) can be administeredthrough intravenous injection, or delivered to a tumor site, andincludes, among other things, a bolus injection. However, the inventionis not limited to this, or any other, method of administration.

A “therapeutic” treatment is a treatment administered to a patient whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs and/or decreasing or diminishing the frequency,duration and intensity of the signs.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

By the term “specifically binds,” as used herein, is meant an antibody,or a ligand, which recognizes and binds with a cognate binding partner(e.g., a stimulatory and/or costimulatory molecule present on a T cell)protein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell (e.g., an aAPC of the invention,among others).

“Loaded” with a peptide, as used herein, refers to presentation of anantigen in the context of an MHC molecule. “Loaded” as used herein alsomeans the binding of an antibody to an Fc binding receptor on a cell,such as CD32 and/or CD64.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible COStimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

“Superagonist antibody,” as used herein, means an antibody thatspecifically binds with a molecule on a T cell and can mediate a primaryactivation signal event in a T cell without interaction of the TCR/CD3complex or CD2 on the T cell. Such superagonist antibody includes, butis not limited to, a superagonist anti-CD3 antibody, a superagonistanti-CD28 antibody, and a superagonist anti-CD2 antibody.

Unless referred to as a “superagonist”, an anti-CD2 antibody, ananti-CD28 antibody, and the like, is a co-stimulatory ligand as definedelsewhere herein, and provides a co-stimulatory signal rather than aprimary activation signal.

To “treat” a disease as the term is used herein, means to reduce thefrequency of the disease or disorder reducing the frequency with which asymptom of the one or more symptoms disease or disorder is experiencedby an animal.

By the term “vaccine” as used herein, is meant a composition, a proteinor a nucleic acid encoding a protein, or an aAPC of the invention, whichserves to protect an animal against a disease and/or to treat an animalalready afflicted with a disease by inducing an immune response,compared with an otherwise identical animal to which the vaccine is notadministered or compared with the animal prior to the administration ofthe vaccine.

“Immunovaccine,” as used herein, means an aAPC that can elicit adetectable immune response when administered to an animal. Morepreferably, an immunovaccine is an aAPC that stimulates and activates Tcells when administered to the animal, such that it generates adetectable T cell immune response to a pathogen, a tumor cell, and thelike, when compared to a T cell the immune response, if any, in anotherwise identical animal to which the immunovaccine is notadministered.

Description

The invention relates to the surprising discovery that a humanerythromyeloid cell line, K562, that does not express MHC class I orclass II molecules, and which was previously believed to be refractoryto genetic manipulation techniques, can be readily transduced usinglentivirus vectors to express numerous molecules, including, but notlimited to, stimulatory ligands, co-stimulatory ligands, antigens (e.g.,tumor, viral, and the like), cytokines, etc.

Further, the data disclosed herein demonstrate that several (at leastnine) exogenous nucleic acids expressing several proteins can be readilyintroduced into and expressed in these cells, but that the level ofexpression of the proteins is higher than that achieved usingplasmid-based expression systems and the expression is stable andcontinues for many months without detectable decrease. In addition, theK562-based artificial antigen presenting cell (aAPC), which does notexpress MHC class I or II molecules, can be transduced with and readilyexpresses them. Remarkably, aAPC transduced with a nucleic acid encodingan antigen of interest processed the antigen and presented it properlyto a T cell thereby producing antigen-specific T cells without need toidentify the epitope recognized by the T cell. Surprisingly, asdemonstrated by the data disclosed herein, the aAPC cell properlyprocessed and presented the antigen.

I. Compositions

The present invention encompasses an isolated artificial antigenpresenting cell (aAPC), where the cell comprises a K562 cell transducedusing a lentiviral vector (LV). Moreover, the LV encodes at least oneimmune stimulatory and co-stimulatory ligand. While the data disclosedherein demonstrate that about nine nucleic acids encoding about ninedifferent molecules transduced into a K562 cell were stably and highlyexpressed in long-term culture, there is nothing to suggest that this isa limit in the number or kinds of molecules that can be introduced intothese cells. Instead, any molecule or ligand, whether stimulatory,co-stimulatory, cytokine, antigen, Fcγ receptor, and the like, can beintroduced into these cells to produce an aAPC of the invention.

The skilled artisan would appreciated, based upon the disclosureprovided herein, that numerous immunoregulatory molecules can be used toproduce an almost limitless variety of aAPCs once armed with theteachings provided herein. That is, there is extensive knowledge in theart regarding the events and molecules involved in activation andinduction of T cell, and treatises discussing T cell mediated immuneresponses, and the factors mediating them, are well-known in the art.Further, the extensive disclosure provided in WO 03/057171 andUS2003/0147869 is incorporated by reference as if set forth in itsentirety herein. More specifically, a primary signal, usually mediatedvia the T cell receptor/CD3 complex on a T cell, initiates the T cellactivation process. Additionally, numerous co-stimulatory moleculespresent on the surface of a T cell are involved in regulating thetransition from resting T cell to cell proliferation. Suchco-stimulatory molecules, also referred to as “co-stimulators”, whichspecifically bind with their respective ligands, include, but are notlimited to, CD28 (which binds with B7-1 [CD80], B7-2 [CD86]), PD-1(which binds with ligands PD-L1 and PD-L2), B7-H3, 4-1BB (binds theligand 4-1BBL), OX40 (binds ligand OX40L), ICOS (binds ligand ICOS-L),and LFA (binds the ligand ICAM). Thus, the primary stimulatory signalmediates T cell stimulation, but the co-stimulatory signal is thenrequired for T cell activation, as demonstrated by proliferation.

Thus, the aAPC of the invention encompasses a cell comprising astimulatory ligand that specifically binds with a TCR/CD3 complex suchthat a primary signal is transduced. Additionally, as would beappreciated by one skilled in the art, based upon the disclosureprovided herein, the aAPC further comprises at least one co-stimulatoryligand that specifically binds with at least one co-stimulatory moleculepresent on a T cell, which co-stimulatory molecule includes, but is notlimited to, CD27, CD28, CD30, CD7, a ligand that specifically binds withCD83, 4-1BB, PD-1, OX40, ICOS, LFA-1, CD30L, NKG2C, B7-H3, MHC class I,BTLA, Toll ligand receptor and LIGHT. This is because, as discussedpreviously and as demonstrated by the data disclosed elsewhere herein, aco-stimulatory signal is required to induce T cell activation andassociated proliferation. Other co-stimulatory ligands are encompassedin the invention, as would be understood by one skilled in the art armedwith the teachings provided herein. Such ligands include, but are notlimited to, a mutant, a variant, a fragment and a homolog of the naturalligands described previously.

These and other ligands are well-known in the art and have been wellcharacterized as described in, e.g., Schwartz et al., 2001, Nature410:604-608; Schwartz et al., 2002, Nature Immunol. 3:427-434; and Zhanget al., 2004, Immunity. 20:337-347. Using the extensive knowledge in theart concerning the ligand, the skilled artisan, armed with the teachingsprovided herein would appreciate that a mutant or variant of the ligandis encompassed in the invention and can be transduced into a cell usinga LV to produce the aAPC of the invention and such mutants and variantsare discussed more fully elsewhere herein. That is, the inventionincludes using a mutant or variant of a ligand of interest and methodsof producing such mutants and variants are well-known in the art and arenot discussed further herein.

Thus, the aAPC of the invention comprises at least one stimulatoryligand and at least one co-stimulatory ligand, such that the aAPC canstimulate and expand a T cell comprising a cognate binding partnerstimulatory molecule that specifically binds with the stimulatory ligandon the aAPC and a cognate binding partner co-stimulatory molecule thatspecifically binds with the co-stimulatory ligand on the aAPC such thatinteraction between the ligands on the aAPC and the correspondingmolecules on the T cell mediate, among other things, T cellproliferation, expansion and immune response as desired. One skilled inthe art would appreciate that where the particular stimulatory andco-stimulatory molecules on a T cell of interest are known, an aAPC ofthe invention can be readily produced to expand that T cell. Conversely,where the stimulatory and co-stimulatory molecules on a T cell ofinterest are not known, a panel of aAPCs of the invention can be used todetermine which molecules, and combinations thereof, can expand that Tcell. Thus, the present invention provides tools for expansion ofdesirable T cells, as well as tools for elucidating the molecules onparticular T cells that mediate T cell activation and proliferation.

The skilled artisan would understand that the nucleic acids of theinvention encompass an RNA or a DNA sequence encoding a protein of theinvention, and any modified forms thereof, including chemicalmodifications of the DNA or RNA which render the nucleotide sequencemore stable when it is cell free or when it is associated with a cell.Chemical modifications of nucleotides may also be used to enhance theefficiency with which a nucleotide sequence is taken up by a cell or theefficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

Further, any number of procedures may be used for the generation ofmutant, derivative or variant forms of a protein of the invention usingrecombinant DNA methodology well known in the art such as, for example,that described in Sambrook and Russell (2001, Molecular Cloning, ALaboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor,N.Y.), and Ausubel et al. (2002, Current Protocols in Molecular Biology,John Wiley & Sons, NY). Procedures for the introduction of amino acidchanges in a protein or polypeptide by altering the DNA sequenceencoding the polypeptide are well known in the art and are alsodescribed in these, and other, treatises.

The invention includes a nucleic acid encoding a costimulatory ligand,or antigen, wherein a nucleic acid encoding a tag polypeptide iscovalently linked thereto. That is, the invention encompasses a chimericnucleic acid wherein the nucleic acid sequences encoding a tagpolypeptide is covalently linked to the nucleic acid encoding at leastone protein of the invention, or biologically active fragment thereof.Such tag polypeptides are well known in the art and include, forinstance, green fluorescent protein (GFP), an influenza virushemagglutinin tag polypeptide, a herpesvirus tag polypeptide, myc,myc-pyruvate kinase (myc-PK), His₆, maltose binding protein (MBP), aFLAG tag polypeptide, and a glutathione-S-transferase (GST) tagpolypeptide. However, the invention should in no way be construed to belimited to the nucleic acids encoding the above-listed tag polypeptides.Rather, any nucleic acid sequence encoding a polypeptide which mayfunction in a manner substantially similar to these tag polypeptidesshould be construed to be included in the present invention.

The nucleic acid comprising a nucleic acid encoding a tag polypeptidecan be used to localize a protein of the invention, or a biologicallyactive fragment thereof, within a cell, a tissue, and/or a wholeorganism (e.g., a human, and the like), and to study the role(s) of theprotein in a cell. Further, addition of a tag polypeptide facilitatesisolation and purification of the “tagged” protein such that theproteins of the invention can be produced and purified readily. Moreimportantly, as demonstrated elsewhere herein, expression of acostimulatory ligand comprising a tag allows the detection of expressionof the ligand, and further permits isolation of cells expressing theligand using many methods, including, but not limited to, cell sorting.

The present invention also provides for analogs of proteins or peptideswhich comprise a costimulatory ligand as disclosed herein. Analogs maydiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both. For example, conservative amino acid changes maybe made, which although they alter the primary sequence of the proteinor peptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

-   -   glycine, alanine;    -   valine, isoleucine, leucine;    -   aspartic acid, glutamic acid;    -   asparagine, glutamine;    -   serine, threonine;    -   lysine, arginine;    -   phenylalanine, tyrosine.        Modifications (which do not normally alter primary sequence)        include in vivo, or in vitro, chemical derivatization of        polypeptides, e.g., acetylation, or carboxylation. Also included        are modifications of glycosylation, e.g., those made by        modifying the glycosylation patterns of a polypeptide during its        synthesis and processing or in further processing steps; e.g.,        by exposing the polypeptide to enzymes which affect        glycosylation, e.g., mammalian glycosylating or deglycosylating        enzymes. Also embraced are sequences which have phosphorylated        amino acid residues, e.g., phosphotyrosine, phosphoserine, or        phosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

The present invention should also be construed to encompass “mutants,”“derivatives,” and “variants” of the peptides of the invention (or ofthe DNA encoding the same) which mutants, derivatives and variants arecostimulatory ligands, cytokines, antigens (e.g., tumor cell, viral, andother antigens), which are altered in one or more amino acids (or, whenreferring to the nucleotide sequence encoding the same, are altered inone or more base pairs) such that the resulting peptide (or DNA) is notidentical to the sequences recited herein, but has the same biologicalproperty as the peptides disclosed herein, in that the peptide hasbiological/biochemical properties of a costimulatory ligand, cytokine,antigen, and the like, of the present invention (e.g., expression by anaAPC where contacting the aAPC expressing the protein with a T cell,mediates proliferation of, or otherwise affects, the T cell).

Among a “biological activity”, as used herein, is included acostimulatory ligand which when transduced into a K562 cell is expressedand, when the cell is contacted with a T cell expressing a cognatecostimulatory molecule on its surface, it mediates activation andstimulation of the T cell, with induced proliferation.

Indeed, the present invention provides a powerful novel screening assayfor the identification of mutants, variants, fragments, and homologs ofcostimulatory ligands in that a potential novel form of a costimulatoryligand can be transduced and expressed in the aAPC of the invention. Theability of the aAPC to stimulate and/or activate a T cell can beassessed and compared with the ability of an aAPC comprising the wildtype or “natural” costimulatory ligand to stimulate and/or activate anotherwise identical T cell. In this way, functional variants,demonstrating the ability to activate/stimulate the T cell to a greater,lesser or equal extent as the control wild type ligand, can be readilyidentified, isolated and characterized. Such novel variants ofcostimulatory ligands are potential research tools for elucidation of Tcell processes, and also provide important potential therapeutics basedon inhibiting or inducing T cell activation/stimulation, such as, butnot limited to, administration of a variant with inhibitory activitywhich can compete with the natural ligand to inhibit unwanted T cellresponses such as, but not limited to, transplant rejection. Conversely,a variant demonstrating greater costimulatory ligand activity can beused to increase a desired T cell response, such as, but not limited to,administration to an immunosuppressed patient. For instance, anexemplary variant ligand can be engineered to be more effective that thenatural ligand or to favor the binding of a positive costimulatorymolecule (CD28) at the expense of a negative regulator (CTLA-4). These,and many other variations are encompassed in the invention.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that a costimulatory ligand encompasses an antibodythat specifically binds with the costimulatory molecule present on a Tcell that the ligand also binds with. That is, the invention encompassesan aAPC comprising not only a costimulatory ligand (e.g., CD80 and CD86,among others) that bind a costimulatory molecule on a T cell (e.g.,CD28), but also encompasses at least one antibody that specificallybinds with the costimulatory molecule (e.g., anti-CD28). Numerousantibodies to the costimulatory molecules are presently available, orthey can be produced following procedures that are well-known in theart.

The skilled artisan would understand, based upon the disclosure providedherein, that an aAPC comprising an antibody can be produced, asexemplified elsewhere herein, by introducing a nucleic acid encodingCD32, the human Fcγ receptor, into the aAPC. Further, as disclosedelsewhere herein, an aAPC that binds an antibody, such as a CD3 antibodyor an CD28 antibody, can be produced by expressing a nucleic acidencoding CD64 on the aAPC. CD64 is the high affinity human FcγRIreceptor. The CD32 and/or CD64 expressed on the aAPC surface can then be“loaded” with any desired antibody that binds with CD32 and/or CD64,including, but not limited to, antibody that specifically binds CD3 andantibody that specifically binds with CD28. Moreover, the inventionencompasses an aAPC wherein a nucleic acid encoding the antibody ligandof interest, perhaps linked to an IRES sequence, is transduced andexpressed on the surface of the aAPC thereby eliminating the need forexpression of CD32 and/or CD64 and loading thereof. Thus, the presentinvention includes an aAPC transduced with a nucleic acid encoding atleast one antibody that specifically binds with CD3, CD28, PD-1, B7-H3,4-1BB, OX40, ICOS, CD30, HLA-DR, MHCII, Toll Ligand Receptor and LFA,among others, as well as an aAPC transduced with CD32 and/or CD64 andloaded with at least one antibody that specifically binds with theafore-mentioned molecules.

Further, the invention encompasses an aAPC wherein the co-stimulatoryligand is a cognate binding partner that specifically binds with aco-stimulatory molecule, as well as where the ligand is an antibody thatspecifically binds with a costimulatory molecule, and any combinationthereof; such that a single aAPC can comprise both nucleic acidsencoding costimulatory ligands and/or antibodies specific forcostimulatory molecules present on the Tcell, and any combinationthereof.

The invention also encompasses an aAPC comprising a nucleic acidencoding an antigen of interest. A wide plethora of antigens areincluded, such as, but not limited to, tumor antigens, e.g., telomerase,melanoma antigen recognized by T cells (MART-1), melanomaantigen-encoding genes, 1, 2, and 3 (MAGE-1, -2, -3), melanoma GP 100,carcinoembryonic antigen (CEA), breast cancer antigen HER-2/Neu, serumprostate specific antigen (PSA), Wilm's Tumor 1 (WT-1), mucin antigens(MUC-1, -2, -3, -4), and B cell lymphoma idiotypes. This is because, asdemonstrated by the data disclosed elsewhere herein, K562-based aAPCcomprising an antigen, can process and present the antigen in thecontext of MHC (where the cell is also transduced with a nucleic acidencoding a MHC class I or class II molecule) thereby producingantigen-specific T cells and expanding a population thereof. The datadisclosed demonstrate that hTERT-specific CTLs were produced byexpanding hTERT+ T cells using an aAPC transduced with CD32 and 4-1BBL(K32/4-1BBL). Thus, aAPCs can be used to expand and produce sufficientantigen specific T cells in order to administer the T cells to a patientin need thereof thus providing an immunovaccine treatment directedagainst tumor cells bearing the antigen. Therefore, an antigen ofinterest can be introduced into an aAPC of the invention, wherein theaAPC then presents the antigen in the context of the MCH Class I or IIcomplex, i.e., the MHC molecule is “loaded” with the antigen, and theaAPC can be used to produce an antigen-specific T cell.

Similarly, a viral, or any other pathogen, antigen can also betransduced and expressed by the aAPC. The data disclosed elsewhereherein demonstrate that matrix protein (flu-MP tetramer) positive Tcells sorted and stimulated irradiated aAPC cells(K2/CD3/4-1BBL/FLU-GFP) loaded with anti-CD28 antibody expanded the Tcells providing large numbers of antigen specific CTLs specific for theviral antigen. These data demonstrate that the aAPCs of the inventioncan be used to expand and produce antigen-specific T cells to be used totreat viral, and other pathogenic, infections.

Additionally, the invention encompasses an aAPC transduced with anucleic acid encoding at least one cytokine, at least one chemokine, orboth. This is because the data disclosed elsewhere herein amplydemonstrate that an aAPC transduced with a nucleic acid encoding aninterleukin (e.g., IL-7, IL-15, and the like) stably expressed theinterleukin. Moreover, using a LV vector comprising an internal ribosomeentry site (IRES), the interleukin can be secreted from the aAPCs (e.g.,a K562 transduced with a LV vector such as, but not limited to, pCLPSCD32-IRES-IL-7, -12, -15, -18, and -21). Other cytokines that can beexpressed by aAPC include, but are not limited to, interferon-γ (IFNγ),tumor necrosis factor-α (TNFα), SLC, IL-2, IL-4, IL-23, IL-27 and thelike. The invention further includes, but is not limited to, chemokineRANTES, MIP-1a, MIP-1b, SDF-1, eotaxin, and the like.

Thus, the invention encompasses a cytokine, including a full-length,fragment, homologue, variant or mutant of the cytokine. A cytokineincludes a protein that is capable of affecting the biological functionof another cell. A biological function affected by a cytokine caninclude, but is not limited to, cell growth, cell differentiation orcell death. Preferably, a cytokine of the present invention is capableof binding to a specific receptor on the surface of a cell, therebyaffecting the biological function of a cell.

A preferred cytokine includes, among others, a hematopoietic growthfactor, an interleukin, an interferon, an immunoglobulin superfamilymolecule, a tumor necrosis factor family molecule and/or a chemokine. Amore preferred cytokine of the invention includes a granulocytemacrophage colony stimulating factor (GM-CSF), tumor necrosis factoralpha (TNFα), tumor necrosis factor beta (TNFβ), macrophage colonystimulating factor (M-CSF), interleukin-1 (IL-1), interleukin-2 (IL-2),interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15),interleukin-21 (IL-21), interferon alpha (IFNα), interferon beta (IFNβ),interferon gamma (IFNγ), and IGIF, among many others.

A chemokine, including a homologue, variant, mutant or fragment thereof,encompasses an alpha-chemokine or a beta-chemokine, including, but notlimited to, a C5a, interleukin-8 (IL-8), monocyte chemotactic protein1alpha (MIP1α), monocyte chemotactic protein 1 beta (MIP1β), monocytechemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 3(MCP-3), platelet activating factor (PAFR),N-formyl-methionyl-leucyl-[³H]phenylalanine (FMLPR), leukotriene B₄(LTB_(4R)), gastrin releasing peptide (GRP), RANTES, eotaxin,lymphotactin, IP10, I-309, ENA78, GCP-2, NAP-2 and/or MGSA/gro. Oneskilled in the art would appreciate, once armed with the teachingsprovided herein, that the invention encompasses a chemokine and acytokine, such as are well-known in the art, as well as any discoveredin the future.

The skilled artisan would appreciate, once armed with the teachingsprovided herein, that the aAPC of the invention is not limited in anyway to any particular antigen, cytokine, costimulatory ligand, antibodythat specifically binds a costimulatory molecule, and the like. Rather,the invention encompasses an aAPC comprising numerous molecules, eitherall expressed under the control of a single promoter/regulatory sequenceor under the control of more than one such sequence. Moreover, theinvention encompasses administration of one or more aAPC of theinvention where the various aAPCs encode different molecules. That is,the various molecules (e.g., costimulatory ligands, antigens, cytokines,and the like) can work in cis (i.e., in the same aAPC and/or encoded bythe same contiguous nucleic acid or on separate nucleic acid moleculeswithin the same aAPC) or in trans (i.e., the various molecules areexpressed by different aAPCs).

In this way, as would be understood by one skilled in the art, basedupon the disclosure provided herein, the dose and timing ofadministration of the aAPCs can be specifically tailored for eachapplication. More specifically, where it is desirable to providestimulation to a T cell using certain molecules expressed by an aAPC, orseveral aAPCs, followed by stimulation using another aAPC, or severalaAPCs, expressing a different, even if overlapping, set of molecules,then a combination of cis and trans approaches can be utilized. Inessence, the aAPCs of the invention, and the methods disclosed herein,provide an almost limitless number of variations and the invention isnot limited in any way to any particular combination or approach. Theskilled artisan, armed with the teachings provided herein and theknowledge available in the art, can readily determine the desiredapproach for each particular T cell. Alternatively, based upon thedisclosure provided herein, which provides methods for assessing theefficacy of the T cell stimulation and expansion methods disclosedherein, the skilled artisan can determine which approach(es) can beapplied to the particular T cells to be expanded or stimulated.

The skilled artisan would understand, based upon the disclosure providedherein, that various combinations of molecules to be expressed in theaAPCs of the invention may be favored. While several of thesecombinations of molecules are indicated throughout the specification,including, but not limited to, the combinations exemplified at Tables 1,2, 3 and 4, the invention is in no way limited to these, or any otheraAPC comprising any particular combination of molecules. Rather, oneskilled in the art would appreciate, based on the teachings providedherein, that a wide variety of combinations of molecules can betransduced into a cell to produce the aAPC of the invention. Themolecules encompass those known in the art, such as those discussedherein, as well as those molecules to be discovered in the future.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising an aAPC of the invention as an activeingredient. Such a pharmaceutical composition may consist of the activeingredient alone, as a combination of at least one active ingredient(e.g., an effective dose of an APC) in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the active ingredient and one or more pharmaceuticallyacceptable carriers, one or more additional (active and/or inactive)ingredients, or some combination of these.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs, birds including commercially relevant birds suchas chickens, ducks, geese, and turkeys, fish including farm-raised fishand aquarium fish, and crustaceans such as farm-raised shellfish.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,intra-lesional, buccal, ophthalmic, intravenous, intra-organ or anotherroute of administration. Other contemplated formulations includeprojected nanoparticles, liposomal preparations, resealed erythrocytescontaining the active ingredient, and immunologically-basedformulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers and AZT, protease inhibitors, reverse transcriptaseinhibitors, interleukin-2, interferons, cytokines, and the like.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

The aAPC of the invention and/or T cells expanded using the aAPC, can beadministered to an animal, preferably a human. When the T cells expandedusing an aAPC of the invention are administered, the amount of cellsadministered can range from about 1 million cells to about 300 billion.Where the aAPCs themselves are administered, either with or without Tcells expanded thereby, they can be administered in an amount rangingfrom about 100,000 to about one billion cells wherein the cells areinfused into the animal, preferably, a human patient in need thereof.While the precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal and the routeof administration.

The aAPC may be administered to an animal as frequently as several timesdaily, or it may be administered less frequently, such as once a day,once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

An aAPC (or cells expanded thereby) may be co-administered with thevarious other compounds (cytokines, chemotherapeutic and/or antiviraldrugs, among many others). Alternatively, the compound(s) may beadministered an hour, a day, a week, a month, or even more, in advanceof the aAPC (or cells expanded thereby), or any permutation thereof.Further, the compound(s) may be administered an hour, a day, a week, oreven more, after administration of aAPC (or cells expanded thereby), orany permutation thereof. The frequency and administration regimen willbe readily apparent to the skilled artisan and will depend upon anynumber of factors such as, but not limited to, the type and severity ofthe disease being treated, the age and health status of the animal, theidentity of the compound or compounds being administered, the route ofadministration of the various compounds and the aAPC (or cells expandedthereby), and the like.

Further, it would be appreciated by one skilled in the art, based uponthe disclosure provided herein, that where the aAPC is to beadministered to a mammal, the cells are treated so that they are in a“state of no growth”; that is, the cells are incapable of dividing whenadministered to a mammal. As disclosed elsewhere herein, the cells canbe irradiated to render them incapable of growth or division onceadministered into a mammal. Other methods, including haptenization(e.g., using dinitrophenyl and other compounds), are known in the artfor rendering cells to be administered, especially to a human, incapableof growth, and these methods are not discussed further herein. Moreover,the safety of administration of aAPC that have been rendered incapableof dividing in vivo has been established in Phase I clinical trialsusing aAPC transfected with plasmid vectors encoding some of themolecules discussed herein.

II. Methods

The invention encompasses a method for specifically inducingproliferation of a T cell expressing a known co-stimulatory molecule.The method comprises contacting a T cell that is to be expanded with anaAPC comprising a lentivirus vector encoding a ligand that specificallybinds with that co-stimulatory molecule. As demonstrated elsewhereherein, contacting a T cell with a K562-based aAPC comprising, amongother things, a costimulatory ligand that specifically binds a cognatecostimulatory molecule expressed on the T cell surface, stimulates the Tcell and induces T cell proliferation such that large numbers ofspecific T cells can be readily produced. The aAPC expands the T cell“specifically” in that only the T cells expressing the particularcostimulatory molecule are expanded by the aAPC. Thus, where the T cellto be expanded is present in a mixture of cells, some or most of whichdo not express the costimulatory molecule, only the T cell of interestwill be induced to proliferate and expand in cell number. The T cell canbe further purified using a wide variety of cell separation andpurification techniques, such as those known in the art and/or describedelsewhere herein.

As would be appreciated by the skilled artisan, based upon thedisclosure provided herein, the T cell of interest need not beidentified or isolated prior to expansion using the aAPC. This isbecause the aAPC is selective and will only expand the T cell(s)expressing the cognate costimulatory molecule.

Preferably, expansion of certain T cells is achieved by using severalaAPCs or a single aAPC, expressing various molecules, including, but notlimited to, an antigen, a cytokine, a costimulatory ligand, an antibodyligand that specifically binds with the costimulatory molecule presenton the T cell. As disclosed elsewhere herein, the aAPC can comprise anucleic acid encoding CD32 and/or CD64 such that the CD32 and/or theCD64 expressed on the aAPC surface can be “loaded” with any antibodydesired so long as they bind CD32 and/or CD64, which are Fcγ receptors.This makes the “off the shelf” aAPC easily tailored to stimulate anydesired T cell.

The invention encompasses a method for specifically inducingproliferation of a T cell expressing a known co-stimulatory molecule.The method comprises contacting a population of T cells comprising atleast one T cell expressing the known co-stimulatory molecule with anaAPC comprising a LV encoding a ligand of the co-stimulatory molecule.As disclosed elsewhere herein, where an aAPC expresses at least oneco-stimulatory ligand that specifically binds with a co-stimulatorymolecule on a T cell, binding of the co-stimulatory molecule with itscognate co-stimulatory ligand induces proliferation of the T cell. Thus,the T cell of interest is induced to proliferate without having to firstpurify the cell from the population of cells. Also, this method providesa rapid assay for determining whether any cells in the population areexpressing a particular costimulatory molecule of interest, sincecontacting the cells with the aAPC will induce proliferation anddetection of the growing cells thereby identifying that a T cellexpressing a costimulatory molecule of interest was present in thesample. In this way, any T cell of interest where at least onecostimulatory molecule on the surface of the cell is known, can beexpanded and isolated.

The invention includes a method for specifically expanding a T cellpopulation subset. More particularly, the method comprises contacting apopulation of T cells comprising at least one T cell of a subset ofinterest with an aAPC capable of expanding that T cell, or at least anaAPC expressing at least one costimulatory ligand that specificallybinds with a cognate costimulatory molecule on the surface of the Tcell. As demonstrated previously elsewhere herein, binding of theco-stimulatory molecule with its binding partner co-stimulatory ligandinduces proliferation of the T cell, thereby specifically expanding a Tcell population subset. One skilled in the art would understand, basedupon the disclosure provided herein, that T cell subsets include Thelper (T_(H1) and T_(H2)) CD4 expressing, cytotoxic T lymphocyte (CTL)(Tc1 or Tc2) T regulatory (T_(REG)), T_(C/S), naïve, memory, centralmemory, effector memory, and γδT cells. Therefore, cell populationsenriched for a particular T cell subset can be readily produced usingthe method of the invention.

The invention also includes a method for identifying a co-stimulatoryligand, or combination thereof, which specifically induces activation ofa T cell subset. Briefly, the method comprises contacting a populationof T cells with an aAPC comprising a LV encoding at least oneco-stimulatory ligand, and comparing the level of proliferation of the Tcell subset contacted with the aAPC with the level of proliferation ofan otherwise identical T cell subset not contacted with the aAPC. Agreater level of proliferation of the T cell subset contacted with theaAPC compared with the level of proliferation of the otherwise identicalT cell subset which was not contacted with the aAPC is an indicationthat at the co-stimulatory ligand specifically induces activation of theT cell subset to which that T cell belongs.

The method permits the identification of a costimulatory ligand thatspecifically expands a T cell subset where it is not previously knownwhich factor(s) expand that T cell subset. The skilled artisan wouldappreciate that in order to minimize the number of screenings, it ispreferable to transduce as many nucleic acids encoding costimulatoryligands such that the number of assays can be reduced. Further, themethod allows, by combining the various proteins (e.g., stimulatoryligand, costimulatory ligand, antigen, cytokine, and the like), toassess which combination(s) of factors will make the most effectiveaAPC, or combination of aAPCs, to expand the T cell subset. In this way,the various requirements for growth and activation for each T cellsubset can be examined.

In one aspect, the method comprises contacting various aAPCs with the Tcell subset without first characterizing the costimulatory molecules onthe surface of the T cell subset. Also, the invention encompasses amethod where the costimulatory molecule(s) present on the surface of theT cell subset are examined prior to contacting the aAPCs with the cell.Thus, the present invention provides a novel assay for determining thegrowth requirements for various T cell subsets.

The invention encompasses a method for inducing a T cell response to anantigen in a mammal. The method comprises administering an aAPC thatspecifically induces proliferation of a T cell specific for the antigen.Once sufficient numbers of antigen-specific T cells are obtained usingthe aAPC to expand the T cell, the antigen-specific T cells so obtainedare administered to the mammal according to the methods disclosedelsewhere herein, thereby inducing a T cell response to the antigen inthe mammal. This is because, as demonstrated by the data disclosedherein, that antigen-specific T cells can be readily produced bystimulating resting T cells using the aAPC of the invention.

The invention encompasses a method for inducing a T cell response to anantigen in a mammal in need thereof, the method comprising obtaining apopulation of cells from the mammal wherein the population comprises Tcells, contacting the T cells with an aAPC presenting the antigen in thecontext of an MHC complex, wherein contacting the T cells with the aAPCinduces proliferation of T cells specific for the antigen. Theantigen-specific T cells are administered to the mammal, therebyinducing a T cell response to the antigen in the mammal in need thereof.As stated previously elsewhere herein, the data disclosed elsewhereamply demonstrate that antigen-specific CTLs can be readily produced bycontacting a T cell with an aAPC wherein the aAPC presents the antigenin the context of an MHC complex. As noted previously elsewhere herein,a wide variety of aAPCs can be used, comprising numerous combinations ofvarious molecules (costimulatory ligands, antibodies, antigens, MHCs,and the like), to determine the optimal method for expanding theantigen-specific T cells for administration to a mammal in need thereof.

III. Kits

The invention includes various kits which comprise an aAPC of theinvention, a nucleic acid encoding various proteins, an antibody thatspecifically binds to a costimulatory molecule on the surface of a Tcell, and/or a nucleic acid encoding the antibody of the invention, anantigen, or an cytokine, an applicator, and instructional materialswhich describe use of the kit to perform the methods of the invention.Although exemplary kits are described below, the contents of otheruseful kits will be apparent to the skilled artisan in light of thepresent disclosure. Each of these kits is included within the invention.

The invention includes a kit for specifically inducing proliferation ofa T cell expressing a known co-stimulatory molecule. This is becausecontacting the T cell with an aAPC, specifically induces proliferationof the T cell. The kit is used pursuant to the methods disclosed in theinvention. Briefly, the kit may be used to administer an aAPC of theinvention to a T cell expressing at least one costimulatory molecule.This is because, as more fully disclosed elsewhere herein, the datadisclosed herein demonstrate that contacting a T cell with an aAPCcomprising a costimulatory ligand that specifically binds with thecognate costimulatory molecule present on the T cell, mediatesstimulation and activation of the T cell. Further, the T cells producedusing this kit can be administered to an animal to achieve therapeuticresults.

The kit further comprises an applicator useful for administering theaAPC to the T cells. The particular applicator included in the kit willdepend on, e.g., the method used to administer the aAPC, as well as theT cells expanded by the aAPC, and such applicators are well-known in theart and may include, among other things, a pipette, a syringe, adropper, and the like. Moreover, the kit comprises an instructionalmaterial for the use of the kit. These instructions simply embody thedisclosure provided herein.

The kit includes a pharmaceutically-acceptable carrier. The compositionis provided in an appropriate amount as set forth elsewhere herein.Further, the route of administration and the frequency of administrationare as previously set forth elsewhere herein.

The kit encompasses an aAPC comprising a wide plethora of molecules,such as, but not limited to, those set forth at Tables 1, 2, 3, and 4,elsewhere herein. However, the skilled artisan armed with the teachingsprovided herein, would readily appreciate that the invention is in noway limited to these, or any other, combination of molecules. Rather,the combinations set forth herein are for illustrative purposes and theyin no way limit the combinations encompassed by the present invention.Further, the kit comprises a kit where each molecule to be transducedinto the aAPC is provided as an isolated nucleic acid encoding amolecule, a vector comprising a nucleic acid encoding a molecule, andany combination thereof, including where at least two molecules areencoded by a contiguous nucleic acid and/or are encoded by the samevector. The routineer would understand that the invention encompasses awide plethora of constructs encoding the molecules of interest to beintroduced into an aAPC of the invention.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

EXAMPLES Example 1 Development of Cell-Based Artificial AntigenPresenting Cells (aAPC) for Adoptive Immunotherapy

It has been demonstrated that the intrinsic growth requirements of CD4and CD8 T cells differ (Deeths et al., 1997, Eur. J. Immunol.27:598-608; Laux et al., 2000, Clin. Immunol. 96:187-197; Foulds et al.,2002, J. Immunol. 168:1528-1532). A cell-based aAPC was designed toenable genetic manipulation of the expression of different costimulatorymolecules in addition to CD28 for the long term growth of CD8 cells. Theculture system was based on the fact that costimulatory signals, inaddition to those provided by CD28, are required for optimal CD8 cellgrowth. The human erythromyeloid CML cell line K562 (Lozzio et al.,1975, Blood 45:321-334) was used as a scaffold for the cellular aAPCs,because this cell line does not express HLA proteins that would promoteallogeneic responses. However, K562 do express ICAM (CD54) and LFA-3(CD58), both of which promote interactions with T cells (FIG. 1). Otheradvantages of using K562 cell include, but are not limited to, the factthat irradiated K562 cells can be introduced in the clinical setting asthese cells are mycoplasma-free, propagate in serum-free medium, and areeasily killed by natural killer (NK) cells. Despite the desirability ofusing K562 to produce such aAPCs, K562 cells have been notoriouslydifficult to transduce (Kahl et al., 2004, J. Virol. 78:1421).Surprisingly, the data disclosed herein demonstrate, for the first time,that K562 cells can be transduced, either serially and/or in parallel,with a wide plethora of exogenous nucleic acids to express a number ofmolecules thereby obtaining a library of aAPCs with desired phenotypes.Such LV-based transduction is performed serially and/or in parallel, andMoFlo is used to clone cells demonstrating a desired phenotype. Further,the data demonstrate that an optimal promoter can be selected andpromoter competition can be assessed and eliminated if necessary ordesired. The library of aAPCs produced using the methods of theinvention are then assessed for biologic function in vivo, using anart-recognized model, such as, but not limited to, a NOD/SCID mousemodel.

With regard to use of K562 to produce aAPCs, the disclosures of U.S.patent application Ser. No. 10/336,135 (now published as U.S. PatentApplication Publication No. US2003/0147869A1) and International PatentApplication No. PCT/US03/00339 (now published as InternationalPublication No. WO 03/057171A2) are incorporated by reference as if setforth in their entirety herein.

Production of Lentiviral Vectors

To circumvent the limitations of previously described transfection-basedapproaches to introduce genes into K562 cells, a series of high-titerlentiviral vectors were used, as disclosed elsewhere herein, to stablyintroduce a wide array of costimulatory ligands and MHC molecules intoK562 cells. This allows for the systematic and rapid production for avariety of aAPCs and allows for the determination for the combination ofcostimulatory molecules that yields the optimal expansion and effectorfunctions to HIV-specific T cells. The data disclosed herein demonstratethis approach.

As a non-limiting example, an aAPC of the present invention can comprisesome or all of the ligands, among other things, described herein. Thesevarious constructs are used to transduce the K562 cells using LVs. Theseare merely exemplary and the invention is not limited to theseconstructs, or any other particular construct, for transduction of theaAPCs of the invention with a known molecule of interest to be expressedin the cells.

CD32: A CD32-comprising LV-transduced aAPC was produced using CD32 (SEQID NO:8) amplified from cDNA prepared from neutrophil RNA. Briefly, theneutrophils were isolated by Ficoll gradient from an apheresis productobtained from a normal, anonymous donor. This PCR product was clonedinto pcDNA3.1 via Kpn I and Not I restriction sites that were added tothe ends of each amplifying primer. This vector was digested with XbaIand Sal I and cloned into pCLPS (Parry et al., 2003, J. Immunol.171:166-174) to create pCLPS CD32. Supernatant containing high titerlentiviral vector was obtained by harvesting transfected 293T cells thathad been transfected using a split genome transfection method asdescribed in Dull et al. (1998, J. Virol. 72:8463-8471) and Parry et al.(2003, J. Immunol. 171:166-174).

IL-7: In one embodiment, an aAPC comprising IL7 was produced using IL-7nucleic acid (SEQ ID NO:9) amplified from cDNA and cloned into pcDNA3.1hygromycin. SOE by PCR (consisting of three separate reactions) usingprimers designed with 5′ CD32 XbaI and 3′ IL-7 SalI was performed.Additional templates used during reaction included CD32-pCDNA3.1 andpCLPS m8h28-IRES-YFP followed by restriction enzyme digestion of the PCRproducts to produce CD32-IRES-IL-7 pCLPS. Lentivirus was made asdescribed previously elsewhere herein.

IL-15: In another embodiment, plasmid pVAX Hum IL-15 (comprising SEQ IDNO:10), which comprises an IgE leader peptide attached to mature IL-15sequence, was used. PCR primers were designed to add MluI and SalIrestriction sites to the plasmid sequence. The PCR product was digestedwith the respective enzymes and was cloned into CD32-IRES-IL-7 pCLPS(which was also cut with MluI and SalI to remove the IL-7 gene).Lentivirus was prepared as described herein.

IL-21: In another embodiment, IL-21 (SEQ ID NO:11) was amplified fromactivated human PBMC and cloned into TOPO 3 vector (Invitrogen,Carlsbad, Calif.). Using BamHI and XhoI, the human IL-21 gene wasexcised from the vector and the insert was then cloned into the firstposition of NKG2D-IRES-DAP12 pCLPS construct (which was produced usingBamHI and XhoI). Human CD32 was amplified from CD32-pCLPS (as describedabove) using PCR primers that flanked the ends comprising MluI and SalIsites. The PCR product was digested with the respective enzymes and wascloned into the second position to create IL21-IRES-CD32 pCLPS.Lentivirus was produced as described previously elsewhere herein.

OX40L: In another embodiment, OX40L (SEQ ID NO:12) was amplified fromcDNA obtained from mature dendritic cells (Schlienger et al., 2000,Blood) and was cloned into pCDNA3.1 hygromycin. Restriction enzyme sitesMluI and SalI were added to the ends of PCR primers and the PCR productwas digested with the respective enzymes and was cloned intoCD32-IRES-IL-7 pCLPS, which was also cut with MluI and SalI to removeIL-7 insert. CD32-IRES-OX40L pCLPS lentiviral vector was made asdescribed previously elsewhere herein.

4-1BBL: In another embodiment, 4-1BBL (SEQ ID NO:13) was amplified fromcDNA obtained from activated B cells which were purified by negativeselection as described previously, and which cells were activated withPMA and ionomycin. The PCR product in which Kpn I and NotI sites wereintroduced at the ends, was cloned into KpnI/Not I digested pcDNA 3.1hygromycin. The vector was digested with XbaI and Sal I and was clonedinto XbaI/Sal I digested pCLPS lentiviral vector. pCLPS 4-1BBLlentiviral vector was made as described previously elsewhere herein.

CD80: In yet another embodiment, CD80 (SEQ ID NO:14) was amplified fromcDNA obtained from immortalized B cell line (Vonderheide et al., 1999,Immunity 10:673-679) using PCR primers that introduced BamHI and SalIsites at the ends of the PCR product. Following digestion with theseenzymes, CD80 was cloned into BamHI/SalI digested pCLPS lentiviralvector. CD80-pCLPS lentiviral vector was produced as describedpreviously elsewhere herein.

CD83: In yet a further embodiment, CD83 (SEQ ID NO:15) was amplifiedfrom cDNA prepared from the Ramesh cell line, using primers to introduceXbaI and XhoI restriction sites to the ends of the PCR product.Following digestion with these enzymes, CD83 PCR product was ligatedinto pCLPS (digested with XbaI/SalI). CD83-pCLPS lentiviral vector wasproduced as described previously elsewhere herein.

CD86: In another embodiment, CD86 (SEQ ID NO:16) was amplified from cDNAobtained from mature dendritic cells (which were prepared as describedin Schlienger et al., 2000, Blood) using PCR primers that had BamHI andNot I restriction sites added to the ends. The PCR product was digestedwith BamHI and Not I and ligated into similarly digested pcDNA3.1hygromycin. This vector was digested with BamHI and Sal I and was clonedinto pCLPS. pCLPS CD86 lentiviral vector was produced as describedpreviously elsewhere herein.

ICOS-L: In another embodiment, ICOS-L (SEQ ID NO:17) was amplified usingPCR primers comprising Kpn I and Not I restriction sites at the ends,using cDNA obtained from dendritic cells. The PCR product was clonedinto KpnI and Not I digested pcDNA3.1 hygromycin. The plasmid wasdigested with BamHI and Xho I to excise the ICOS-L insert which wascloned into pCLPS (digested with BamHI/XhoI) to generate ICOS-L-pCLPS.Lentiviral vector was produced as described previously elsewhere herein.

HLA-A*0201: In yet another embodiment, HLA-A*0201 cDNA clone wasobtained from The International Cell and Gene Bank, which is publiclyavailable from the website of the International HistocompatibilityWorking Group organization (ihwg.org) at the cell and genebank sharedresources (cbankover). HLA-A*0201 was amplified by PCR using primerscomprising Bam HI and Sal restriction sites at the ends and theamplification product was cloned into pCLPS. pCLPS HLA-A2 lentiviralvector was produced as described previously elsewhere herein.

Flu-GFP: In one embodiment, a Flu-GFP fusion vector comprising theentire enhanced Green Fluorescent Protein (BD Biosciences, Palo Alto,Calif.) coding region fused to the Flu Matrix Protein 1 nucleotides113-290 (SEQ ID NO:18) was used. This construct was digested with BamHIand Xho I and cloned into Bam HI and Sal I digested pCLPS. pCLPS GFP-flulentiviral vector was produced as described previously elsewhere herein.

DRα: In another embodiment, DRα (SEQ ID NO:19) and DRB4 (SEQ ID NO:20)were cloned using cDNA obtained from CD3/28-activated CD4 T cells usingstandard techniques, and each nucleic acid was cloned into pCLPS. Toproduce K562 cells that expressed DR4, both vectors were simultaneouslytransduced into K562 and HLA-DR cells and cells expressing DR4 wereisolated using flow cytometry as described elsewhere herein.

In addition, ILT3 (SEQ ID NO: 21) and ILT4 (SEQ ID NO:22) were expressedin the aAPC of the present invention essentially according to methodsdisclosed elsewhere herein and known in the art.

High-titer, high efficiency, third-generation lentiviral vectors (LV)were used to efficiently produce aAPC. These vectors have a number ofbuilt-in safety features that make them ideally suited for humantherapeutics. Specifically, approximately 90% of the HIV-1 sequenceshave been removed from the transfer vector leaving only the packagingand integration sequences physically linked to the payload gene.Replication-incompetent packaged LVs are generated using a split genomeapproach. Specifically, 293 T cells are transfected with four separateplasmids encoding HIV gag/pol, VSV G protein (env), HIV rev, and thetransfer vector. Lentiviral vectors were produced after transfection of293T HEK cells cultured in RPMI 1640 (BioWhittaker, Inc. Rockville Md.),10% FCS, 2 mM glutamine and 100 IU/mL penicillin, 100 ug/mLstreptomycin. Cells were seeded at 5×10⁶ per T 150 tissue culture flask24 hours prior to transfection. All plasmid DNA was double purifiedusing a CsCl gradient. Cells were transfected with 7 μg pMDG.1 (VSV-Genvelope), 18 μg pRSV.rev (HIV-1 Rev encoding plasmid), 18 μgpMDLg/p.RRE (packaging plasmid) and 15 μg pCLS transfer plasmid usingFugene 6 (Roche Molecular Biochemicals, Indianapolis, Ind.). Media waschanged 6 hours after transfection and the viral supernatant washarvested at 24 hours and 48 hours post-transfection. Viral particleswere concentrated 10-fold by ultra centrifugation for 3 hours at 28,000RPM with a Beckmann SW28 rotor as described in Reiser (2000, Gene Ther.7:910-913).

As a result of this strategy, three independent and highly unlikelyrecombination events would have to occur to create areplication-competent vector. As an additional safety precaution, thisvector was rendered self-inactivating by deleting the 3′LTR promoter(Zufferey et al., 1998, J. Virol. 72:9873-9880). Thus, upon integrationthe only functioning promoter is the supplied internal promoter (in thiscase CMV) juxtaposed with the payload gene and thus, no HIV sequencesare transcribed.

Using this lentiviral vector transduction approach, several high titerlentiviral vectors have been created for CD83 and ICOS-L andKA2/32/86/4-1BBL aAPCs and the parent KA2/32/86 has been created (FIG.3), as well as other aAPCs described elsewhere herein. Briefly, K562cells were transduced with lentiviral expression vectors encoding CD32,HLA-A2, 4-1BBL and an influenza MP1GFP fusion protein, sorted for singleclones expressing all four markers, and expanded for four weeks (FIG.3). In addition, a wide variety of lentiviral vectors, comprisingnumerous combinations of molecules useful for transduction of K562-basedaAPCs, have been produced (P) or have been designed (D), as set forth inTable 1.

TABLE 1 pCLPS CD32 (P) pCLPS siRNA-PD-L1 (D) pCLPS CD32/IRES/GM-GCSF (D)pCLPS siRNA-B7-H3 (D) pCLPS CD32/IRES/IL-7 (P) pCLPS siRNA-TGFbeta (D)pCLPS CD32/IRES/IL-12 (D) pCLPS IDO (D) pCLPS CD32/IRES/IL-15 (P) pCLPSGFP-flu matrix (P) pCLPS CD32/IRES/IL-18 (D) pCLPS GFP/IRES/pol (P)pCLPS CD32/IRES/IL-21 (P) PCLPS HLA DR0101 (D) pCLPSCD32/IRES/Interferon alpha (D) pCLPS HLA A201 (P) pCLPS CD32/SLC (D)pCLPS ICOSL (P) pCLPS CD30L (D) pCLPS CD86 (P) pCLPS OX40L (P) pCLPSCD83 (P) pCLPS 4-1BBL (P) pCLPS CD80 (P) pCLPS GITRL (D) pCLPS CD70 (D)pCLPS CD40 (D)K562 Cells

K562 cells were isolated from a patient with chronic myelogenousleukemia in terminal blast crisis (Lozzio et al., 1975, Blood45:321-334). K562 may represent a DC precursor that does not express MHCmolecules or T cell costimulatory ligands, but retains many otherattributes that make DCs effective APCs, such as, but not limited to,cytokine production, adhesion molecule expression, and macropinocytosis.These attributes may be unique to K562 cells, as the monocytic cell lineU-937 was unable to function as an effective aAPC. Thus, K562 cellsrepresent ideal scaffolds onto which the desired MHC molecules andcostimulatory ligands can be introduced to establish a DC-like aAPC.Such an aAPC has all the advantages of DCs, including high levels of MHCexpression, a wide array of costimulatory ligands, and the ability toengage in cytokine crosstalk with a T cell. K562-based aAPCs also lackthe disadvantages of DCs, such as their limited life span, lack ofreplicative capacity, and ill defined maturation requirements (Lee etal., 2002, Vaccine 20:A8-A22).

Transduction of K562 Cells to Produce aAPCs

The data disclosed herein demonstrate the creation of a K562 aAPC vialentivirus-mediated introduction of costimulatory ligands that enableK562 cells to better mimic the potent T cell stimulatory ability of DCs.

K562 cells were transfected with the human Fc receptor CD32 (“K32 cell”)to permit loading with anti-CD3 and anti-CD28 antibodies, and the cellwas also transfected with human 4-1BBL (“K32/4-1BBL cells”) for addedco-stimulation (FIG. 1). KA2/32/86/4-1BBL/CD83 aAPCs were produced witha CD83 lentiviral vector by spinoculation (0′Doherty et al., 2000, J.Virol. 74:10074-10080) to transduce the KA2/32/86/4-1BBL parent.Approximately 5 million KA2/32/86/4-1BBL cells were mixed with 500 μl ofconcentrated virus (5×10⁷-5×10⁸ IFU/ml) and spun at 1200 g for 2 hours.Five days post transduction the cells were stained with a CD83 specificAb and a Moflo sorter was used to isolate high expressing clones. 15-20days post sorting, colonies of single clones were visible and thesecolonies of single clones were screened by CD83 expression. Highexpressors were expanded further and the expression levels of the otherintroduced markers (HLA-A2, 4-1BBL, CD86 and CD32) was measured toensure that the descendants are similar to parent cell line in all butCD83 expression. The K32/86/4-1BBL/ICOS-L and K32/86/4-1BBL/ICOS-L/CD83aAPCs were created in this manner using the appropriate viruses.

Using the methods described herein, stable expression of of at leastnine (9) genes has been accomplished in a K562 aAPC. The following geneswere transduced into a K562 cell and were stably expressed, as detectedusing flow cytometry: Flu-GFP (FIG. 8A); CD80 (FIG. 8B); CD86 (FIG. 8C);4-1BBL (FIG. 8D); and HLA ABC (FIG. 8E). TheKT32/A2/4-1BBL/40L/CD80/CD83/CD86 also stably expressed detectablelevels of CD32, CD83, CD40L, and ICOS-L. These expression levelsremained constant for greater than 3 months of continuous culturewithout any selection. In addition, the production of several aAPCs isillustrated in FIG. 3. These aAPCs comprise expression of all of thetransgenes driven by the CMV promoter. Although diminishing expressionlevels of transgenes due to sequestration of CMV-specific transcriptionfactors could occur, (Cahill et al., 1994, FEBS Lett. 344:105-108; Kanget al., 1992, Science 256:1452-1456) to date no evidence of any problemswith serially transducing K562 cells with five different lentiviralvectors has been detected (FIG. 3).

Using the methods described above for transducing a K562 cell, parentalK562cc was compared to LV-transduced K562 cells (e.g., transduced withand expressing five and eight genes). As illustrated in FIGS. 7 and 8,LV transduced cells exhibit favorable growth kinetics compared withotherwise identical, but non-transduced, parental cells.

In addition to the expression of costimulatory ligands, the aAPCs of theinvention can be used to produce various cytokines, as exemplified bythe production of IL-7 and IL-15 by K562 cells transduced withCD32/IRES/IL-7 and CD32/IRES/IL-15 vectors. The cells were sorted forhigh CD32 expression and production of the respective interleukin wasassessed (FIG. 13), demonstrating that the appropriate cytokine wasproduced.

Further, using the methods for transducing a K562 cell described above,aAPCs express CD32 from an LV at a greater level than the expression ofCD32 in an otherwise identical K562 cell transfected using a plasmidvector (FIG. 11). These data demonstrate that, surprisingly, K562 wereeasily transduced using lentiviral vectors. FIG. 11D is a graphdepicting that expression of CD32 using a LV to transduce K562 cells isgreater than the level of CD32 expression in an otherwise identical K562cell transfected using a plasmid vector. Moreover, the data disclosedherein demonstrate that the level of CD32 expression was maintained forgreater than nine months. Moreover, this level of CD32 expression wasmaintained for greater than nine months. The characterization of aAPCcells expressing CD64 is described below.

In addition, the data disclosed herein demonstrate that the aAPCs growin culture in medium free of fetal calf serum (FCS), an importantconsideration for production of aAPCs for use in treatment of humanpatients (see FIG. 7). These data demonstrate that the novel aAPCs ofthe invention grow in defined medium (Aim V) comprising 3% AB serum.That is, various K562-based aAPCs were produced by transducing parentalK562 (k562cc) using lentivirus vectors (“LV”): KT32 (1 gene);KT32/4-1BBL/CD86 (3 genes); KT32/4-1BBL/CD86/A2/Flu-GFP (5 genes). Inaddition, as illustrated in FIG. 7, introduction of a lentiviral vectordoes not significantly alter the growth rates of the K562 cells. Thesedata demonstrate that master cell banks can be produced usingLV-transduced K562 aAPCs and that aAPCs grow as well as the parentalcells.

The present data has demonstrated the methods for transducing K562 cellsand the expression and growth properties of these aAPCs. In addition,the long-term stablility and sufficient expression of acytokine/costimulatory molecule transduced into a K562-based aAPC hasbeen evaluated. CD32 has been stably expressed in a transduced K562 cellfor longer than nine months. Further, detectable and stable expressionof at least eight exogenous molecules introduced into a K562-based aAPChas also been achieved (KT32-A2-41 BBL-40L-80-83-86), and there is nodata to suggest that additional molecules will not be similarlyexpressed. Indeed, an aAPC has been produced expressed nine genes(including ICOS-L) for greater than 60 days at this time. Thus, atpresent, the ability of the aAPCs of the invention to express a varietyof molecules in a single aAPC is not limited. Further, the aAPCs of theinvention are negative for mycoplasma and replication competentlentivirus (RCL), and their safety and lack of any contaminatingpathogens can be readily assessed.

The present invention comprises numerous K562-based aAPCs produced,according to the methods set forth herein, including, but not limited tothose set forth in Table 2. These aAPCs, comprising combinations ofvarious immunostimulatory molecules, can be used for both ex vivo and invivo methods comprising expansion of certain T cell subsets,identification of combinations of factors that expand T cell subsets, aswell as cell based and gene therapy where the aAPCs, and or T cellsexpanded thereby, are administered to a patient in need thereof. Ofcourse, the present invention is not limited to these, or any particularaAPCs, and the list set out in Table 2 is merely illustrative of theteachings provided herein.

TABLE 2 Polyclonal T cell expanding Antigen-specific T cell expansionaAPCs (designated “KT32” aAPCs (designated “KTA2” series) series) KT32KTA2 KT32/4-1BBL KTA2/86 KT86 ktA2-86-ICOSL KT83 ktA2-41BBL KT80ktA2-41BBL-FLU-GFP KT86-80 ktA2-41BBL-86-FLU-GFP KT83-80ktA2-32-41BBL-FLU-GFP KT32/86/83 ktA2-86-FLU-GFP-CD40L kt32-ICOSLktA2-41BBL-86-FLU-GFP-83 kt86-ICOSL ktA2-41BBL-86-FLU-GFP-CD40Lkt32-41BBL-80 ktA2-86-FLU-GFP-CD40L kt32-41BBL-86 (hi, lo)*ktA2-41BBL-86-83-40L-80-Flu-GFP kt32-41BBL-83 kt32-IL7 kt32-IL15ktIL-21 kt32-41BBL-86-83 kt32-41BBL-86-83-IL15 kt32-CD30L kt32-OX40Lkt32-HLA-DR (MHC class II)

Example 2 In Vivo Therapeutic Use of aAPCs

The invention includes LV-engineered K562 aAPC for in vivo therapeuticvaccination and for the ex vivo expansion of T cells for therapeuticuses. The antigens, cytokines, and/or costimulatory molecules can betransduced into a K562 cell under the control of the same or separatepromoters/regulatory sequences. Further, a nucleic acid encoding thetumor cell antigen can be transduced into the cell or the antigen can beotherwise loaded into the cell such that the cell processes and presentsthe appropriate epitope in the context of an MHC protein. This isbecause it has been demonstrated elsewhere herein that K562 cells havethe ability to process and present antigens without the need to firstidentify or isolate the specific antigen or epitope required. Thus, acell extract (comprising at least one membrane component of a tumorcell) can be loaded into the K562-based aAPC and the natural ability ofthe cell to process and present the relevant antigen is exploited. Whilecustomized aAPCs are set forth herein, these are for illustrativepurposes only, and the invention is not limited to these embodiments setforth in Table 3. This is because, as would be appreciated by theskilled artisan armed with the teachings provided elsewhere herein, awide plethora of molecules can be transduced and expressed by the aAPCsin virtually limitless combinations.

TABLE 3 Autoimmune/ Epithelial Heme Skin Transplantation (breast/colon/Malignancy (Mela- (RS/SLE/ Indica- lung/ovary/ (CML/AML/ noma/GVHD/Organ tion prostate) Lymphoma/ALL) Merkel) Txp) aAPC KT-BCLOPKT-CALA KT-MM KT-T_(REG) costim CD80/83/ CD80/83/ CD80/83/ CD86 dim/41BBL/ 41BBL/ 41BBL/ B7H-3/ OX40L OX40L OX40L MHCII antigens gp100/MAGETo be determined To be selected cyto- GM-CSF/ GM-CSF/ GM-CSF/TGFbeta/IL-10 kines IL-15/SLC IL-15/SLC IL-15/SLC

Example 3 Ex Vivo Therapeutic Uses of aAPCs

Other aAPCs can be prepared for ex vivo use, such as, but not limitedto, adoptive immunotherapy and gene therapy. Among the customizedversions of aAPC for such ex vivo uses are, inter alia, the constructsdisclosed in Table 4 below. That is, T cells isolated from a subject canbe stimulated and expanded in vitro using these, or a wide plethora ofother, aAPCs then the T cells can be introduced into the subject therebyproviding adoptive immunotherapy thereto. Additionally, the expanded Tcells can be genetically engineered to express an exogenous protein thatwas not expressed, or was expressed at a lower level, compared withexpression of the protein in the T cell prior to, or in the absence of,the genetic engineering. Thus, the present invention provides both exvivo cell based adoptive immunotherapy and gene therapy using the aAPCsof the invention to expand T cells used for autologous transplantationof a subject in need thereof. Table 4 merely sets forth severalillustrative examples of aAPCs that can be used for such cell/genetherapy, but the invention is not limited to these exemplary “off theshelf” aAPCs of the invention.

TABLE 4 CTLs Genetically engineered Indication (melanoma, HIV, RCC) Tcells (HIV/cancer) AAPC KT-A2 KT32 Costim CD80/83/83/4-1BBL Anti-CD3,28/4- 1BBL/83) Antigens Of choice None cytokines IL-7/IL-15/SLC IL-7,IL-15

Example 4 Stimulation of Human CD 4 T Cells with aAPCs

The data disclosed herein demonstrate that long-term growth of CD4 Tcells was obtained using K32/CD3/28 and K32/86CD3 aAPCs in the absenceof exogenous cytokines, but U937-based aAPCs were not effective (FIG.5). Moreover, the data demonstrate that K562-based aAPCs (e.g.,K32/CD3/28, K32/86/CD3) mediate long-term growth of CD4 T cells moreeffectively bead-based aAPCs (CD3/28 coated beads) where both beads andcells are loaded with CD3 and CD28. These results demonstrate thatdetectable “cross-talk” occurs between the K562-based aAPCs and T cellswhich is not possible using bead-based systems. As illustrated in FIG.5, not all tumor cell lines have the capacity to serve as artificialAPCs and the data further demonstrate the ability of K562 cells to serveas potent APCs, which was unexpected. These surprising results supportthe significant improvement over prior art methods that is possibleusing K562-based aAPCs.

The data disclosed herein further demonstrate the usefulness ofK562-based aAPCs for inducing cytokine and/or costimulatory moleculeexpression by CD4 T cells (FIG. 6A-6D). These data all demonstrate thatK562-based aAPCs are far superior than U937-based aAPCs in inducingcytokine and costimulatory (costim) gene expression by T cells and thatsome aAPC constructs are better than others, somewhat depending on thecytokine and/or costimulatory molecule being expressed. Morespecifically, while K32/CD3/28 was generally superior compared with theother aAPCs, expression of B7-H3 was actually greater by K32/CD3 in thepresence of CD4 when compared with K32/CD3/28 under similar conditions.These data demonstrate that K562 aAPCs upon interacting with T cells,produce a series of additional cytokines and costimulatory molecules(APC and T cell cross talk) that can further enhance T cell activationand expansion. More specifically, various K562- and U937-based aAPCstransduced with various vectors encoding certain molecules (e.g.,K32/CD3/28, K32/CD3, K32, U32/CD3/28, U32/CD3, U32) were assayed fortheir ability to induce expression of molecules of interest (e.g.,IL-15, PD-L-1, PD-L2, and B7-H3). The data disclosed herein demonstratethat K562-based aAPCs induced detectable expression of these moleculesand did so to far greater extent than U937-based cells. These datafurther demonstrate the usefulness of the of novel aAPCs of theinvention, and the significant improvement over prior art methods sincethese data all demonstrate that K562-based aAPCs are far superior thanU937-based aAPCs in inducing cytokine and costimulatory (costim) geneexpression by T cells. These results are particularly remarkable giventhe previous teachings demonstrating that K562 parental cell linedemonstrates poor T cell stimulatory activity (Britten et al., 2002, J.Immunol. Methods 259:95-110).

Given the superior results of aAPCs of the present invention incomparison to parental K562 cells, U-937 based aAPCs and beads, therelative abilities of the various aAPCs disclosed herein was evaluated.Some aAPC constructs are better than others at mediating an effect uponcertain T cells, and that the effectiveness varies somewhat depending onthe cytokine and/or costimulatory molecule, or combinations thereof,being expressed. More specifically, while K32/CD3/28 was generallysuperior compared with the other aAPCs, expression of B7-H3 was actuallygreater by K32/CD3 in the presence of CD4 when compared with K32/CD3/28under similar conditions. These data further demonstrate that certaincombinations of molecules expressed in the novel K562-based aAPCs have agreater effect on certain populations of T cells. These data thereforeprovide a novel system for assessing the effectiveness of variouscombinations of molecules to achieve desired effect(s) and/or tostimulate and expand T cell subsets of interest none of which werepossible prior to the present invention.

Example 5 Stimulation of Human CD8 T Cells with aAPCs

Briefly, 50,000 irradiated KT32/4-1BBL/CD86 were coated with anti-CD3 Aband were mixed with 100,000 freshly isolated CD8 T cells from a healthydonor. Every 10-12 days the CD8 T cells were re-stimulated with freshlyirradiated KT32/4-1BBL/CD86 aAPCs. The total number of cells that wouldhave accumulated if no cells have been discarded is depicted as asemi-log plot of total cell number versus days in culture (FIG. 9). Asalso illustrated in FIG. 9, polyclonal CD8 T cells expanded by aAPCstransduced with CD32 and 4-1BBL (K32/4-1BBL) expanded 18,600 fold after43 days.

The data disclosed herein further demonstrate the expansion of antigenspecific CD8 T_(CM) using an aAPC (e.g., K32/K-41BBL aAPC). Briefly,both flu tet+ and flu tet− T cells were expanded (FIG. 10A-10C) as afunction of days in culture wherein interleukin 2 (IL-2) was added tothe culture medium on day 20. The data demonstrate a shift by day 16 incells stained for CD8 expression using flow cytometry where the cellswere sorted as being Flu tet− or Flu-tet+, demonstrating antigenspecific proliferation of CD8 T_(CM) cells cultured with K32/4-1BBLaAPC. At day 26, the cells were assayed for their ability tospecifically lyse tet+ or tet− cells that were T2-null or expressedT2-flu. The data demonstrate that cell killing was specific for ter+,T2-flu target cells as demonstrated by a chromium release assay (FIG.10E). The percent specific lysis was a function of the effector:target(E:T) cell ratio, with maximum specific cell lysis detected at a 10:1ratio, and decreasing thereafter as the E:T ration was decreased to 1:1.At all E:T ratios examined, cell lysis specific for tet+, T2-flu wasobserved using the cells expanded using the aAPC.

To determine if the aAPCs of the present invention can act in anantigen/MHC-specific manner, K562 cells were transduced with HLA-A2,CD86, 4-1BBL, CD32, CD64 and influenza MP1 mini gene that encodes the A2restricted epitope GILGFVFTL (SEQ ID NO:1) linked to GFP. FACS analysisof this KA2/32/86/4-1BBL/Flu GFP aAPC demonstrated that all five markersare expressed at high levels (FIGS. 3 and 12), and stable expression ofall the transgenes was observed for as long as nine months of continuousculture.

To demonstrate that these aAPCs were sufficient to expand antigenspecific cells, 1500 flu tetramer positive cells were isolated from aHLA-A2 donor and were mixed with 3000 irradiated KA2/32/86/4-1BBL/FluGFP aAPCs. Every 10 days freshly irradiated KA2/32/86/4-1BBL Flu GFPaAPCs are added to the culture so that there would be approximately 1aAPC for every two T cells. After 22 days, there were approximately 10million CD8 T cells. These cells were stained with a Flu specific A2tetramer and greater than 90% of the cells were Flu specific (FIG. 12F),which was approximately 250-fold enrichment compared with the pre-sortcells (FIG. 12E). These data demonstrate that K562 cells have theability to process and present antigen and expand antigen specific Tcells without the use of an antibody. Importantly, KA2/32/86/4-1BBL/FluGFP aAPCs were unable to expand T cells from the tetramer negativefraction of cells.

A similar experimental protocol was used to expand flu specific T cells,but anti-CD3 was used to deliver signal “one” rather than a peptidebound by MHC class I (FIG. 2). Briefly, the cells were stained withA*0201 tetrameric MHC loaded influenza matrix protein peptide amino acidsequence (GILGFVTVL; SEQ ID NO:1), and sorted into positive and negativefractions. After 17 days of expansion using K32/4-1BBL/CD3/28 aAPC, eachpopulation of cells was stained with the same tetramer used for theinitial sorting. Only about 60% of the cells were flu tetramer positiveand the overall level of staining was lower. This suggests that theKA2/32/86/4-1BBL/Flu GFP aAPCs selectively expand a T-cell receptor(TCR) that has the highest affinity for the GILGFVFTL (SEQ ID NO:1)peptide presented by the HLA-A2.

To determine whether the K32/4-1BBL aAPC coated with anti-CD3 and CD28Abs could be used to expand antigen-specific CD8 T cells, a populationof MHC tetramer-sorted primary CD8⁺ T cells were cultured withK32/4-1BBL/CD3/28 aAPCs for 10 weeks (FIG. 2A). T cells from A*0201individuals with immunity to influenza were stained with anti-CD8 mAband an A*0201 MHC tetramer complexed to an A*0201-restricted peptideepitope of the influenza matrix protein (flu MP tetramer). Thelow-frequency (less than about 0.1%) tetramer⁺ population was sorted andstimulated with irradiated K32/4-1BBL aAPCs coated with anti-CD3 andCD28 Abs. All cells were re-stimulated with K32/4-1BBL aAPCs at about 10day intervals. No specific flu stimulation was provided during culture.Exponential growth curves were obtained for several months of culture.In a representative experiment, approximately 8,000 antigen-specific Tcells yielded 1.5×10⁹ cells after one month of culture (FIG. 2B), whichis a number sufficient for effective immunotherapy (Riddell et al.,1995, Annu. Rev. Immunol. 13:545-586). Phenotypic analysis of culturesdemonstrated that the irradiated aAPCs mixed with resting human T cellsyielded a population of pure T cells within one week. Furthermore, fluMP tetramer positive cells displayed potent cytotoxicity for Flu-MPpeptide pulsed T2 targets (FIG. 2C). This strategy can be adapted toexpand HIV-specific CD8 T cells and to use these aAPCs to expand CD8 Tcells with a broad specificity.

The K32/4-1BBL aAPC was also used to expand hTERT specific cytotoxiclymphocytes (CTL). hTERT-specific CTLs expanded using a K32/4-1BBL aAPCspecifically lysed carcinoma cells expressing HLA-A2 and telomerase⁺(OV-7) but not carcinoma cells that are telomerase⁺ and HLA-A2⁻(SK-OV-3) (FIG. 10E). Thus, the aAPC induced expansion of antigenspecific CTLs that require the antigen to be recognized in the contextof HLA-A2. Further, during expansion, the CTLs, which were obtained froma breast cancer patient vaccinated with hTERT, demonstrated a detectableincrease, as assessed using MoFlo sorting, in the percentage of tet+ CD8CTLs during expansion by K32/4-1BBL aAPC. The timing of the MoFlosorting corresponding to each FIG. 10A-10C is indicated on the graphshowing population doublings as indicated by an arrow (FIG. 10D).

Surprisingly, the data disclosed herein demonstrate, for the first time,that K562-based aAPC have the ability to process an antigen which isthen presented to T cells thereby expanding antigen-specific T cellswhere the particular epitope responsible for expansion is not known apriori. More specifically, purified T cells were obtained from an HLAA*0201 donor and the cells were stained with anti-CD8 mAb and an A*0201MHC tetramer complexed with an A*0201 restricted epitope of theinfluenza matrix protein (flu-MP-tetramer). The tetramer positivepopulation of approximately 1,500 cells, was sorted and stimulated usingirradiated KTA2/CD32/4-1BBL/FLU-GFP aAPCs loaded with anti-CD28antibody. The cells were re-stimulated with KTA2/CD32/4-1BBL/FLU-GFPaAPCs at approximately every 10-12 days. Interleukin-2 was added to theculture at every feeding, approximately every 2-3 days.

After twenty-six days of culture, most of the T cells were flu-MPtetramer positive compared with the initial pre-sort population,demonstrating that the aAPC processed and presented the flu-specificantigen and efficiently expanded flu-specific CTLs (FIG. 12A-12F). Theseresults demonstrate that the aAPCs of the invention can be used toexpand and produce antigen-specific T cells even where the preciseepitope of the antigen required to produce the cells is not known. Thisis important to the development of transfer therapy where the preciseantigen that stimulates a T cell is not known. This is the case for,among other things, tumor specific antigens, where very few are known.Thus, the present invention relates to providing a pathogen (e.g., avirus), or other molecule to which a specific T cell response isdesired, to a K562 cell and allowing the cell to process and present theantigen thereby generating the desired antigen-specific response.

In an additional experiment, it was demonstrated that specific ligandspromote unexpected expansion of antigen specific CD8 T cells. FIG. 21illustrates CMV specific T cells isolated by tetramer sorting andstained with CFSE and mixed with the aAPC at 2:1 ratio. FIG. 21Aillustrates the CMV specific CD8 cells, FIG. 21B illustrates CMVspecific CD8 cells contacted with K32 cells loaded with anti-CD3antibody. FIG. 21C depicts CMV specific CD8 cells contacted with aAPCsexpressing CD32, IL-15, 4-1BBL, CD80 and anti-CD3. These datademonstrate that the addition of costimulatory ligands (in this caseCD80 IL-15 and 4-1BBL) unexpectedly promote the expansion of antigenspecific CD8 T cells.

Example 6 aAPC Expansion of HIV-1 Specific CD8 T Cells with RestoredEffector Functions

Attempts to augment HIV-specific T cell response by autologous transferof antigen-specific CD8 T cells have not resulted in long-termcontainment of HIV infection (Tan et al., 1999, Blood 93:1506; Koenig etal., 1995, Nature Med. 1:330-336; Brodie et al., 1999, Nature Med.5:34-41; Riddell et al., 1996, Nature Med. 2:216-223; Lieberman et al.,1997, Blood 90:2196-2206). The inability of these cells to survive invivo precluded any attempt to measure an anti-HIV response in aclinically meaningful way. In some cases the early demise of these cellswas easily explained as immune recognition of a selectable marker(Riddell et al., 1996, Nature Med. 2:216-223). In other cases, thereasons for T cell death upon infusion were less clear. These earlytrials used slightly different variations of loading peripheralmononuclear blood cells (PMBCs) or lymphoblastoid cell lines (LCLs) withHIV-specific peptides, performing limiting dilution to isolate clones,and expanding the T cells ex vivo for several months using high levelsof exogenous IL-2 and TCR triggering in the absence of costimulation toproduce up to 1×10⁹ HIV-specific T cells. Without wishing to be bound byany particular theory, it may be that extended ex vivo culture, coupledwith a dependence on high levels of IL-2, led to the initiation ofapoptosis in these cells once infused back into their host.

While bead-based aAPCs (CD3/28 coated beads) are efficient vehicles toexpand CD4 T cells from HIV-infected individuals (Levine et al., 1996,Science 272:1939-1943), there are a number of potential advantages tousing cell-based aAPC (gene-modified K562 cells) for use in HIV adoptivetransfer clinical trials. First, cell-based aAPCs expand T cells muchmore robustly than bead-based systems (Parry et al., 2003, J. Immunol.171:166-174). This reduces the time required to obtain therapeuticquantities of T cells, lowering the cost of these therapies and perhapsimproving the function of the cells once they are infused back into thepatient. Next, additional costimulatory molecules can easily beintroduced into the aAPC by lentiviral transduction. Importantly, CD3/28coated beads are effective only to expand CD4 T cells (Laux et al.,2000, Clin. Immunol. 96:187-197; Deeths et al., 1999, J. Immunol.163:102-110). Thus, in order to test the immune reconstitution potentialof infusing both CD4 and CD8 ex vivo expanded T cells back into HIVinfected individuals, new expansion systems must be developed and suchsystems are disclosed herein.

It is useful to create APCs that optimally expand CD8 T cells from HIVinfected individuals. Previously, K562 cells were transfected with CD32(to bind stimulatory Ab) and 4-1BBL as the minimal aAPC that induceslong-term expansion of CD8 T cells. Also, it was demonstrated that CD86triggered CD28 endowed T cells with the same proliferative ability astriggering CD28 endowed T cells with an anti-CD28 Ab (Thomas et al.,2002, Clin. Immunol. 105:259-272). Because it was desire to develop Abindependent culture systems, CD86 was used rather than anti-CD28 totrigger both CD28 costimulatory effects on T cell expansion and HIVreplication. Thus, the following five aAPCs can be used to expand andfunctionally test CD8 T cells from HIV infected patients: KA2/32/86,KA2/32/86/4-1BBL, KA2/32/86/4-1BBL/CD83, KA2/32/86/4-1BBL/ICOS-L andKA2/32/86/4-1BBL/CD83/ICOS-L. KA2/32/86 can stimulate CD8 T cells butdoes not endow them with long term growth potential (Maus et al., 2002,Nature Biotechnol. 20:143-148). This aAPC serves as a negative (orbaseline) control to which other costimulatory ligands can be compared.All of the aAPCs created express both HLA-A2 and CD32. This allows useof the same aAPC to expand polyclonal CD8 T cells by having signal onedelivered by CD32 bound anti-CD3 Ab (FIG. 1) or antigen specific T cellsby having signal one initiated by peptide bound in HLA-A2.

KA2/32/86/4-1BBL is the minimal aAPC to expand CD8 T cells from healthydonors (Maus et al., 2002, Nature Biotechnol. 20:143-148). Additionalcostimulatory signals may be required to expand HIV-specific T cellswith improved effector functions. Comparison of cells expanded with thisaAPC with those cells expanded with the aAPCs listed below allows forthe identification of any additional desirable costimulation signals.

KA2/32/86/4-1BBL/CD83 is used because CD83 is a marker of mature DCswhose role in T cell activation has recently been investigated.Stimulation of T cells with magnetic beads coated with anti-CD3 andCD83Ig fusion protein enhanced the ratio of CD8 to CD4 T cells,suggesting that CD83 ligation preferentially activates CD8 T cells.Moreover, CD83-expressing tumor cells were more efficiently killed byCD8 T cells and primed the immune system to also reject CD83-deficienttumors (Scholler et al., 2001, J. Immunol. 166:3865-3872; Scholler etal., 2002, J. Immunol. 168:2599-2602).

KA2/32/86/4-1BBL/ICOS-L is used because ICOS-L binds the CD28-relatedmolecule inducible costimulator protein (ICOS), delivering a potentcostimulatory signal to T cells that enhances production of effectorcytokines (IFN-γ, IL-4, and IL-13) but is curiously unable to producehigh levels of IL-2 (Hutloff et al., 1999, Nature 397:263-266) or inducethe survival factor Bcl-xL (Parry et al., 2003, J. Immunol.171:166-174). The precise roles that ICOS and CD28 play in the immunesystem are still unclear but comparing the outcome of ICOS and CD28blockade in several disease models has revealed clues. Blockade ofeither ICOS or CD28 interferes with both IFN-γ production and generationof protective immunity in lymphocytic choriomeningitis virus (LCMV)(Kopf et al., 2000, J. Exp. Med. 192:53-61) and Toxoplasma gondii(Villegas et al., 2002, J. Immunol. 169:937-943) infection models,suggesting a non-redundant relationship between ICOS and CD28costimulation. Moreover, examination of when the costimulatory blockadeis administered has revealed that CD28 is crucial for priming, whileICOS is more important to maintaining a T cell response (Gonzalo et al.,2001, Nature Immunol. 2:597-604; Coyle et al., 2000, Immunity13:95-105). ICOS-L stimulation promotes effector functions in both CD4and CD8 T cells (Villegas et al., 2002, J. Immunol. 169:937-943;Mittrucker et al., 2002, J. Immunol. 169:5813-5817; Wallin et al., 2001,J. Immunol. 167:132-139).

KA2/32/86/4-1BBL/CD83/ICOS-L allows, among other things, examination ofwhether there is synergy between CD83 and ICOS-L signaling in generatingan immune response.

These aAPCs allow the experimentation of whether the effector functionscan be restored to T cells from HIV-1 infected donors through optimal exvivo expansion. Upon completion of creating the aAPCs, their ability toexpand polyclonal CD8 T cells isolated from HIV infected patients wereassessed and it was determined if and how ex vivo expansion had alteredthe ability HIV-specific T cells to respond to antigen stimulation.Next, a similar analysis using Pol-specific cells isolated from HIVinfected and non infected individuals was preformed. These studiesdemonstrate the potential use of aAPCs for clinical trials and allowsthe study of how HIV infection influences the development of HIVspecific CD8 T cells.

Methods describe in this Example relate to the purification ofPol-specific and polyclonal CD8 T cells from HIV infected andnon-infected individuals. These cells serve as the source material usedin methods disclosed herein.

Source and Purification of HIV-1 Infected T Cells

HLA-A2 donors are used because of the high prevalence of this allele inthe population. Initially, viral phenotype is not used as a patientselection criteria for HLA-A2 donors. It has been demonstrated thatnaïve CD8 T cells express low amounts of CD4 on their cell surface afteractivation, making them susceptible to HIV infection (Yang et al., 1998,J. Exp. Med. 187:1139-1144; Kitchen et al., 1998, J. Virol.72:9054-9060; Flamand et al., 1998, Proc. Natl: Acad. Sci. U.S.A95:3111-3116; Imlach et al., 2001, J. Virol. 75:11555-11564). However,only naïve CD8 T cells appear to have this plasticity and thePol-specific T cells used are by definition memory T cells. Thus, notwishing to be bound to any particular theory, these cells are notinfected and will not become infected upon ex vivo expansion.

In addition to using HLA-2 donors, PBMCs can be used. PBMCs are stainedwith FITC labeled HLA-A2 specific Ab, BB7.2, (BD Pharmingen). Desiredcells can be obtained from HLA-A2 donors apheresis. A cross-section ofCD4 T counts and viral loads is obtained from the apheresis product. Asample of the apheresis product is used to perform a Ficoll/Hypaquegradient, and PBMCs are frozen in 50 million/vial aliquots. With theremaining apheresis product, monocytes are removed by elutriation tocreate PBLs and approximately 50 million CD4 T cells are isolated bynegative selection (Maus et al., 2002, Nature Biotechnol. 20:143-148).The remainder of the PBLs is used to make purified CD8 T cells (bynegative selection) and used in the methods and experiments describedherein.

It has been measured that the percent of HIV Pol-specific T cells bytetramer stain is found to be approximately 0.7%±1.1 (Sun et al., 2003,Journal of immunological methods 272:23-34; Kostense et al., 2002, Blood99:2505-2511; Rinaldo et al., 2000, J. Virol. 74:4127-4138). Thus, from100 million CD8 T cells, about 700,000 HIV Pol-specific CD8 T cells isrecovered. Live sorting HIV infected T cells presents a number oftechnical and safety issues. Vantage SE/DiVa is a full-featured sortercapable of measuring 12 colors plus forward and side scatter. VantageSE/DiVa has been outfitted with enhanced safety features (Perfetto etal., 2003, Cytometry 52A:122-130) that allows it to safely sortinfectious materials into 4 populations at once.

Source and Purification of Pol-Specific T Cells from Non HIV InfectedHost

Seven HLA-A2 breast cancer patients were vaccinated to the Pol peptide,ILKEPVHGV (SEQ ID NO:3), as a control arm to an hTERT peptide vaccine(Vonderheide, 2004, Clin. Cancer Res. 10:828-839). Cells from thevaccinated HLA-A2 breast cancer patients are used to expand Pol-specificcells from a HIV negative host. Frozen PMBCs from these patients areused to purify Pol-specific T cells. The frequency of these Pol-specificT cells is lower than the frequency expected from HIV infected donors(approximately 0.1%) so fewer (but still a sufficient number) of thesecells are obtained to initiate experiments. Pol specific T cellspatients can be obtained from any patient that has been vaccinated witha Pol peptide.

During the ex vivo expansion, the cultures are monitored for theevidence of HIV infection by using p24 ELISA, as HIV infection skews theresults. In the case where an infection is observed, patients who areinfected with R5 viruses are used. R5 viruses are unable to replicate inCD3/28 costimulated T cells due to high levels of secretion of the CCR5natural ligands RANTES, MIP-1 and MIP-1β as well as downregulating thesteady state levels of the CCR5 transcript (Riley et al., 1997, J.Immunol. 158:5545-5553; Carroll et al., 1997, Science 276:273-276).Thus, CD28 costimulation permits the long term growth of R5 infected Tcells without the addition of anti-viral components which may alter theexpansion properties of T cells. Viral tropism is determined using theGHOST cell assay developed by Littman and colleagues.

Also, the experiments disclosed herein do not require pure Pol-specificT cells. HIV-1 Pol-specific CD8 T cells isolated by tetramer coatedmagnetic beads can be used (Maus et al., 2003, Clin. Immunol.106:16-22). This method can provide sufficient enrichment ofPol-specific T cells, and can be used to isolate the Pol-specific cells.

Example 7 Characterization of Ex Vivo Expanded Polyclonal CD8 T Cells

The ability to expand bulk T cells from HIV infected patients isexamined before characterizing the ability of these aAPCs to expand HIVspecific T cells. This allows for the measurement of the expansion rateto determine whether a particular T cell subset is preferentiallyexpanded by one aAPC over another. In addition, the analysis of tetramerstaining coupled with IFN-γ secretion and perforin expression is used todetermine whether a particular aAPC preferentially expands and/or endowsimproved effector function to flu, CMV, EBV or HIV specific CD8 T cells.

A comparison of these studies with those that expand HIV specific CD8 Tcells in isolation to determine whether HIV specific T cells have uniquecostimulatory ligand requirements for expansion and induction ofeffector functions. The following attributes are measured:

T Cell Expansion

To generate therapeutic levels of HIV specific CD8 T cells, T cells areexpanded to between about 10,000 and about 1,000,000 fold (about 13-20population doublings) (Riddell et al., 1995, Annu. Rev. Immunol.13:545-586). There is an inverse relationship between the amount of timeHIV specific CD8 T cells spend in ex vivo culture and the potentialclinical benefit these cells provide to HIV infected patients. Thus, theaAPC which most rapidly expands CD8 T cells from HIV infected patientsis determined and used in the methods disclosed herein.

T Cell Survival and Replicative Potential

The fitness of T cells after ex vivo expansion is an excellentpredicator of their ability to function in vivo. The ability of theseaAPCs to induce the key cell survival gene Bcl-xL is also measured. Thepercentage of apoptotic cells in a culture during the expansion processis used to determine whether any of the cell based aAPCs confer aparticular survival advantage to the expanded T cells. Additionally, thetelomere length of cells after ex vivo expansion is measured todetermine if a particular aAPC is more effective in preserving thereplicative potential of the cells it expands. At the end of eachchromosome there are a large number TTAGGG nucleotide repeats that arecalled telomeres. Each time a cell divides it losses a portion of itstelomeres. Since most cells do not express the enzyme telomerase, whichcan restore copies of the DNA repeat to the ends of chromosomes, it isbelieved that once a cell has lost a critical mass of its telemetriclength it loses its ability to divide. Telomere lengths have been usedin the art as a way to gauge how many times a cell has replicated and,by inference, to assess its future replicative potential (Palmer et al.,1997, J. Exp. Med. 185:1381-1386; Weng et al., 1997, J. Immunol.158:3215-3220). However, T lymphocytes are one of the few cell typesthat can induce telomerase activity (Weng et al., 1996, J. Exp. Med.183:2471-2479) and thus the relative differences between T cellsexpanded using different methods reflect both the number of T cellmitotic events as well as the extent telomerase was induced. Infusingcells that have the most replicative potential is paramount to ensurethat adoptive transferred HIV specific T cells control HIV infection ona long-term basis.

Cytokine Production

Cytokines are important effector molecules and provide insight into Tcell differentiation. The ability of each of the aAPC to induce thefollowing cytokines from CD8 T cells derived from HIV infectedindividuals is quantitated: IL-2 (a key T cell growth factor for ex vivoexpansion and a cell's ability to induce IL-2 correlates well with itslong term growth potential); IL-4 (a marker for TH2 differentiation);and IL-10 (an immunosuppressive cytokine that may be surrogate for Tregulatory cell outgrowth). For HIV infected patients, aAPCs that induceT cells to produce low levels of IL-1.0 is preferred. Other cytokinesinclude, but are not limited to, TGF-β (for the same rationale asIL-10); IFN-γ (a marker for TH1 differentiation and an importanteffector cytokine); and TNFα (an important effector cytokine).

Tetramer and Effector Function Analysis

The tetramer/intracellular IFN-γ and perforin staining assay developedby Immunomics (per manufacturer's instructions) allows both thedetection of antigen specific T cells coupled with phenotypic analysisand functional assays. This flow based method is the most rigorous assayof antigen specific T cell function currently available. This assay isused to determine how ex vivo expansion affects the total number andfunction of HIV specific T cells.

T Cell Expansion

To evaluate how these aAPCs expand bulk HIV-1 infected T cells, eachaAPC was irradiated, coated with anti-CD3 Ab, and mixed with purifiedCD8 T cells from an HIV-1 infected patient at a 1:2 aAPC to T cellratio. To compare the initial rate of cell expansion, the cells weresubject to CFSE staining rather than ³H thymidine uptake to determinehow well each aAPC induced the proliferation of all T cells because CFSEstaining provides a much more quantitative endpoint and allowssimultaneous phenotyping of the expanded cells. Approximately 20 millionpurified CD8 T cells from an HIV infected individual are mixed with 3 μMCFSE for 8 minutes, washed extensively to remove the unbound CFSE, andstimulated with the aAPCs. Every day after stimulation, an aliquot ofcells is removed from each culture and analyzed by flow cytometry. CFSEstaining makes cells highly fluorescent. Upon cell division, thefluorescence is halved and thus the more times a cell divides the lessfluorescent it becomes. The ability of each aAPC to induce T cellproliferation is quantitated by measuring the number of cells thatdivided once, twice, three times and so on. The aAPC that induces themost number of cell divisions at a particular time point is deemed asthe most potent expander of CD8 T cells from HIV infected individuals(Wells et al., 1997, J. Clin. Invest. 100:3173-3183).

However, CFSE staining can only detect a limited number of T celldivisions (approximately 7), and to generate therapeutic quantities of Tcells for immunotherapy, 13-20 population doublings may be necessary.Therefore, to determine how well these aAPCs promote long-term growth ofT cells, cell growth curves are generated. These experiments are set upexactly as the CFSE experiments as described elsewhere herein, but noCFSE is used. Every 2-3 days of culture, T cells are removed from therespective cultures and counted using a Coulter counter which measureshow many cells are present and the mean volume of the cells. The meancell volume is the best predicator of when to restimulate the cells. Ingeneral, when T cells are properly stimulated they triple their cellvolume. When this volume is reduced to more than about half of theinitial blast, it may be necessary to restimulate the T cells tomaintain a log linear expansion (Levine et al., 1996, Science272:1939-1943; Levine et al., 1997, J. Immunol. 159:5921-5930). The timeit takes each aAPC to induce 20 population doublings is calculated. Therelative differences of each aAPC to induce this level of T cellexpansion is an important criteria on which a particular aAPC is used tomove forward to clinical trials.

The phenotypes of the cells expanded by each aAPC are characterized todetermine whether a particular subset is preferentially expanded. Priorto each restimulation, a phenotype analysis of the expanding T cellpopulations is performed to define the differentiation state of theexpanded T cells using the CD27 and CD28 definitions proposed by Appayet al. (2002, Nature Med. 8, 379-385) and CCR7 definitions proposed bySallusto et al. (1999, Nature 401:708-712). Perforin and Granzyme Bintracellular staining are used to perform a gross measure to estimatecytolytic potential.

Apoptosis Rate and Telomere Length

Annexin V/To-Pro (Molecular Probes, Eugene, Oreg.) staining is performedbefore each restimulation to determine whether differences in the growthrate reflect differences in the number of cells undergoing apoptosis.The experimental details of this assay are described in detail in Mauset al. (2002, Nature Biotechnol. 20:143-148). Culture conditions thatlead to the least amount of apoptosis are desirable.

Telomere length is measured using various established techniques knownin the art, but a preferred method is to use the flow FISH method sinceit can be performed relatively quickly using far fewer cells and iseasier to quantitate. In this method, approximately 1 million T cells(although far fewer are required) are denatured using heat and 75%formamide, and then hybridized with a FITC conjugated DNA probe to theTTAGGG sequence. Unbound probe is washed away and the DNA iscounterstained with LDS 751. A mixture of 4 populations of FITC labeledbeads, each having known amounts of molecule equivalents of solublefluorochrome (MESF), is analyzed in each experiment to allow for thecreation of a calibration curve and the determination of the relativetelomere length of each culture over time (Baerlocher et al., 2002,Cytometry 47:89-99). Relative telomere length is measured prior to eachrestimulation and is recorded for whether any of the aAPCs expand Tcells that have significantly longer telomeres.

Cytokine and Bcl-xL Expression

To investigate cytokine production and Bcl-xL expression levels, RNA isisolated from approximately 1 million cells 24 hours after eachstimulation and subjected to quantitative RT-PCR to examine the relativeexpression of but not limited to IL-2, IL-4, IL-10, IFN-γ, TNF-α, andBcl-xL. Experimental details of these established assays can be found inMaus et al. (2002, Nature Biotechnol. 20:143-148), Thomas et al. (2002,Clin. Immunol. 105:259-272), and Parry et al. (2003, J. Immunol.171:166-174). Many discrepancies between TGF-α mRNA levels and secretedcytokine have been noted (Assoian et al., 1987, Proc. Natl. Acad. Sci.USA 84:6020-6024) so TGF-α production is measured by ELISA.

Tetramer and ELISPOT Analysis

The ability of expanded CD8 T cells to recognize common recall antigensand HIV is compared. Prior to expansion, the purified CD8 T cells arestained using the following, among others, HLA-A2 tetramers GLCTLVAML(SEQ ID NO:4) (EBV BMLF), NLVPMVATV (SEQ ID NO:5) (CMV p65), SLYNTVATL(SEQ ID NO:6) (HIV gag p17), ILKEPVHGV (SEQ ID NO:3) (HIV RT pol),GILGFVFTL (SEQ ID NO:1) (Flu matrix), and LLFGYPVYV (SEQ ID NO:7) (HTLVTax) and the frequency of these tetramers is determined prior to ex vivoexpansion. Next, the bulk CD8 T cells from an HIV infected donor isstimulated using the aAPCs disclosed herein and the cells are expandedusing methods described herein. Prior to each restimulation, theexpanded T cells are subjected to tetramer staining to determine whetherthe relative frequency of EBV, CMV and HIV specific T cells has beenaltered by stimulation by a particular aAPC.

It is important to determine the frequency of cells that secrete IFN-γand the frequency of cells that express high levels of perforin afterantigen recognition. The tetramer/intracellular IFN-γ staining assaydeveloped by Immunomics, which combines an intracellular cytokine assaywith tetramer staining to perform flow based functional assays usingantigen specific cells, can be used. The assay can include incorporationof an intracellular stain for perforin expression. Prior to freezing thePBMCs isolated from each patient, a tetramer/intracellular IFN-γ andperforin staining assay using each of the peptides listed elsewhereherein is used to determine the initial tetramer positive/IFN-γsecreting/perforin producing population. For each peptide with acorresponding tetramer, approximately 1 million PBMCs are placed intothree tubes for the following experimental conditions and controls: 1)non-peptide stimulated control, 2) control tetramer stain withoutpeptide stimulation, and 3) tetramer plus peptide tube. Next, theappropriate tetramer is added to each tube and incubated for 30 minutesat room temperature. Two micrograms of peptide is added to the thirdtube and the sample is incubated for 1 hour at 37° C. Brefeldin A isadded to all three tubes and the tubes are incubated for another 4hours. Since all of the tetramers are labeled with P5-Cy5.5 and APC-Cy7,PerCP, and APC labeled CD27 and CD28 Ab can be used to determine thedifferentiation state of the virus specific T cells. The cells arelysed, fixed and permeabelized and IFN-γ FITC labeling protocols areperformed using methods known in the art and methods disclosed herein.There has been success in measuring perforin expression by co-incubatinga PE labeled anti-perforin Ab with the IFN-γ Ab, allowing thesimultaneous measurement of IFN-γ expression and perforin expression.Given the discrepancy reported between IFN-γ secreting HIV specific Tcells and those that have potential to kill (Zhang et al., 2003, Blood101:226-235) this analysis is used to determine how ex vivo expansionwith the various cell based aAPCs alters either of these functionalattributes. The cells are fixed in PFA and analyzed by flow cytometry.This analysis establishes the baseline phenotype of IFN-γ perforinexpressing cells. To determine if the percentage of tetramer positivecells or the phenotype of the cells secreting IFN-γ, or expressing highlevels of perforin, is altered after ex vivo expansion, thetetramer/intracellular IFN-γ staining assay is performed using thedifferentially expanded CD8 T cells. To do this, autologous PBMCs areused and the percentage of CD8 T cells present is determined and the CD8T cells are removed by magnetic bead depletion. The depleted CD8 T cellsare reconstituted with those expanded by the K562 based aAPCs describedelsewhere herein and subjected to the tetramer/intracellular IFN-γstaining assay as described elsewhere herein. These experiments providean indication as to which aAPCs were able to alter the functionalphenotype of HIV specific T cells during ex vivo expansion.

The data disclosed herein demonstrate the phenotype of CD8 T cellsisolated from HIV infected individuals expanded by the cell based aAPCs.While not wishing to be bound to any particular theory, it predictedthat a subset of aAPCs can expand HIV specific T cells with improvedeffector functions. This result would confirm that these effectorfunctions can be restored by ex vivo expansion. By process ofelimination, which signals are necessary for this transformation isdetermined according to the methods described herein. This finding isconfirmed by growing HIV specific T cells in isolation and provides arationale for making the aAPC a GMP reagent and performing Phase Iclinical trial to see if the improved ex vivo expanded CD8 T effectorcells can help control HIV infection in patients. Additionally, thisoutcome establishes an experimental system to study the mechanismsmediating the defect(s) of HIV specific T cells and how a particularcostimulatory ligand can overcome or reverse this defect.

Example 8 Characterization of Ex vivo Expanded Pol-Specific T Cells

The methods disclosed herein provide important insights into which aAPCis best at expanding HIV specific CD8 T cells by examining the expansionand function of these cells within the milieu of polyclonal T cellexpansion. However, the translational value of ex vivo expandedpolyclonal T cells from HIV infected individuals is low since the numberof total CD8 T cells is increased in most HIV infected patients ashomeostatic mechanisms adjust for the loss of CD4 T cells and thereappear to be no gross abnormalities in non HIV specific CD8 T cells(Gandhi et al., 2002, Annu. Rev. Med. 53:149-172). Thus, to improve HIVspecific CD8 T cell response by autologous adoptive transfer, systemsthat expand only the HIV specific T cells and endow them with effectorfunctions that can eliminate HIV infection on a long-term basis isdesired.

It is desirable to expand tetramer isolated Pol-specific T cells usingeach of the cell based aAPCs and determine which generates Pol-specificT cells that best can kill HIV infected cells with the highestreplicative and survival potential. These studies are performed usingPol-specific cells isolated from HIV infected individual with Polspecific T cells isolated from cancer patients vaccinated withPol-specific peptide. This comparison provides a unique insight on theeffects HIV has on the generation of antigen specific cells since thesame reagents and assays can be used to study these Pol-specific T cellsisolated from these two disease types. These studies may provide insightinto the nature of the defect of HIV specific T cells and lead newhypothese{grave over (s)} on how to overcome these defects.

T Cell Expansion and Phenotype

On average, 700,000 Pol-specific T cells are isolated from an HLA-A2positive HIV infected individual and approximately 100,000 Pol-specificT cells from a vaccinated cancer patient. Antigen specific expansion canbe accomplished using as few as 1,500 T cells (FIG. 3). To make theculture conditions equivalent, cultures are started by mixing 7,500Pol-specific T cells and 15,000 of the aAPCs described elsewhere herein(it has been observed that inverting the T cell to APC ratio isimportant when expanding so few cells) with 0.5 ng/ml of anti-CD3 Ab ina total of 100 μl of media in a 96 well plate. To compare the ability ofanti-CD3 and K562 processed and presented antigen to stimulate thesecells, an identical set of cultures whose aAPCs have also beentransduced with a Pol-GFP expression vector is expanded. Thus, 10cultures for each donor's cells are available. Freshly irradiated aAPCsare added to the growing polyclonal T cells every 10-12 days at anestimated 1 aAPC to every 2 T cell ratio. Once the population expands toa point where an accurate quantitation of the number of cells presentcan be calculated using a Coulter counter, the expansion rate of eachpopulation is tracked. The CD28/CD27 phenotype of the Pol-specific Tcells isolated from the HIV and cancer patients before and after T cellexpansion is compared.

Cytokine Production, Bcl-xL Expression, Apoptosis Rate, and TelomereLength Assessment

These studies are performed using methods disclosed elsewhere hereinexcept the analysis is comprised of using several million cells present(approximately 30 days). Nonetheless these studies should confirm theresults disclosed elsewhere herein using polyclonal T cells.

Killing of HIV Infected Targets

One advantage of expanding antigen specific T cells in isolation is thatmultiple killing and other functional assays can be performed in a veryquantitative manner. The first test is the tetramer/intracellular IFN-γand perforin expression assay. As disclosed elsewhere herein, ex vivoexpanded CD8 T cells are mixed with the autologous CD8 depleted PBMCs.Since most of the CD8 T cells are tetramer positive, quantitative datais obtained concerning which aAPCs produce T cells that produce thehighest levels of IFN-γ and perforin upon contact with Pol peptide.Additionally, since the number of tetramer cells is not limiting, acomplete phenotype analysis of these cells using CCR7, CD27, CD28,CD62L, CD45 RO, CD45 RA, and CD57 (Brenchley et al., 2003, Blood101:2711-2720) is performed, thereby correlating the effectorfunction(s) with T cell phenotype.

The ability of the differentially expanded Pol-specific T cells to killT2 cells loaded with the Pol peptide via ⁵¹Cr release assay is assessed.In this assay, the T2 cells are loaded with the Pol peptide ILKEPVHGVprior to the uptake of ⁵¹Cr. After extensive washing, the labeled T2cells are incubated with antigen specific T cells at 1:30, 1:10, 1:3 and1:1 and 1:3 ratios for 4 hours. If the antigen specific T cellsrecognize the peptide presented on the T2 cells and have the ability tokill these cells, then ⁵¹Cr is released and can be detected. No peptidecontrols and detergent lysis controls allow the determination ofspecific lysis. The lowest target to effector ratio in which a highdegree of specific lysis is observed indicates which aAPC expands Tcells with the greatest killing ability.

The ability of ex vivo expanded Pol specific T cells to kill T2 cellsare compared with the ability of these cells to kill Pol expressing CD4T cells. This scenario more closely mimics the in vivo targets of the exvivo expanded CD8 T cells. The ability of these cells to kill HIVinfected CD4 T cells is the ultimate proof of principle. Moreover, it islikely that cells that are infected at this level would generate a veryhigh background in the ⁵¹Cr release assay making it difficult todetermine the effectiveness of the expanded CD8 T cells. As analternative, autologous CD4 T cells are transduce with Pol IRES GFPexpression lentiviral vector that allows the tracking of Pol expressingcells by GFP expression. It was observed that approximate 50% of T cellstransduced by these vectors (see Parry et al., 2003, J. Immunol.171:166-174, for details how the T cells are transduced with lentiviralvectors) which produces enough targets for ⁵¹Cr release assays. Poltransduced as well as non-transduced CD4 T cells are labeled with ⁵¹Cr.CD4 T cells do not uptake ⁵¹Cr as well as T2 cells, so the target isshifted to effector ratio of 1:100, 1:30, 1:10 and 1:3 to improve thesensitivity of the assay. Differences between the non-expanded andexpanded CD8 T cells in their ability to kill Pol expressing CD4 T cellsprovide a strong rationale to further develop one aAPC over another.

CD28 expression is required for long-term ex vivo expansion of CD8 Tcells. It has been recently observed that restoration of CD28 expressionby retroviral transduction (Topp et al., 2003, J. Exp. Med. 198:947-955)restores the ability of CD28 negative cells to produce IL-2 and toexpand without the presence of CD4 T cells. Recently, it has been shownthat IL-12 restores CD28 expression to CD28 negative T cells (Warringtonet al., 2003, Blood 101:3543-3549). To determine whether 1L-12 canenhance CD28 expression and improve the long-term growth potential ofPol-specific T cells, 10 ng/ml of IL-12 is added to the variousaAPCs/Pol-specific cultures and the cells are assessed for how well CD28expression is improved in the Pol-specific cells and whether any of theaAPCs can expand Pol-specific cells in long term culture. Othercytokines, such as IL-21 and IL-15, may work in concert with IL-12 toinduce HIV specific T cell expansion. IL-21 is a multifunctionalcytokine that induces T and B cell proliferation and natural killer (NK)cell differentiation (Parrish-Novak et al., 2002, J. Leukoc. Biol.72:856-863). IL-21 is produced exclusively by activated CD4 T cells andsynergizes with IL-2 and IL-15 to promote CD8 T cell growth. Likewise,IL-15 is a key CD8 T cell survival factor and is produced by activatedmacrophages and DCs (Waldmann et al., 2001, Immunity 14:105-110) whoseaddition may also enhance CD8 T cell expansion.

In the event cytokines fail to permit long term expansion of HIVspecific T cells, Pol-specific T cells are transduced with a lentivirusthat expresses CD28. As outlined in Parry et al., 2003, J. Immunol.171:166-174, greater than 90% of T cells can be transduced with a singletransgene vector (as disclosed herein, IRES containing vectors are lessefficient). Thus, to test whether transduction of CD28 restoreslong-term growth to Pol-specific T cells, Pol-specific T cells arespinoculated (O'Doherty et al., 2000, J. Virol. 74:10074-10080) withCD28 expressing lentiviral vector immediately prior to mixing thesecells with aAPCs. T cell activation induced by these aAPCs facilitatesintegration of this vector and expression of the transgene can beobserved 12 hours post transduction. These cells are cultured asdisclosed elsewhere herein, and Pol specific T cells that are recovered.This is an indication that CD28 costimulation is required for the longterm expansion of these cells. If lentiviral transduction is used as anecessary step to expand HIV specific T cells, it may slow thetranslational impact of these results. However, it has been recentlydemonstrated that lentiviral transduction of T cells from a HIV infectedindividual may be a viable therapeutic option.

Example 9 Expansion of HIV Specific CD8 T Cells with Broad Specificity

Infusion of a single CD8 clone that recognized a Nef epitope led to theselection of viruses that did not express this epitope (Koenig et al.,1995, Nature Med. 1:330-336) indicating that T cells with multiplespecificities are required to prevent HIV escape mutants. A potentiallypowerful way to generate T cells that recognize multiple epitopes of aspecific virus from a patient is to have the cell-based aAPCs uptake andpresent antigen similar to that of natural APCs. By loading a patient'schemically inactivated virus onto an MHC-expressing, K562-based aAPC andmixing in autologous T cells, patient-specific anti-HIV T cells can beexpanded. By providing this optimal situation by which HIV specific Tcells encounter HIV specific antigens, T cells that recognize bothimmunodominant and cryptic antigens can be expanded, thus resulting in agreater potential for controlling HIV infection. Moreover, T cells froma non-infected, healthy person with shared HLA class I alleles can be exvivo vaccinated with the recipient's chemically inactivated virus;expanded and infused into the HIV infected patient. This represents apowerful potential treatment option for an individual with advanceddisease and with a limited T cell repertoire.

Recently, Lu et al. (2003, Nature Med. 9:27-32), demonstrated thatmacaques infused with DCs loaded with a chemically inactivated form ofSIV had significantly less viral RNA and DNA levels, suggesting that theT cell response initiated using this immunotherapy approach can controlSIV infection. These data suggest that properly primed T cells cancontrol SIV infection and strengthens the prospects for a cell-based HIVvaccine in humans (Bhardwaj et al., 2003, Nature Med. 9:13-14).Inactivated virus is used to load MHC-expressing, K562-based aAPCssimilarly to the chemically inactivated SIV study described elsewhereherein. The complexes are used to expand HIV-specific T cells ex vivo tocreate a patient specific T cell therapeutic vaccine. The complexes canalso be administered to a patient using an in vivo approach.

CD107a and 107b are lysosomal associated proteins that are not normallyfound on the T cell surface. Upon TCR triggering, degranulation of CD8 Tcells occurs rapidly, and CD107 and other lysosomal proteins aretransported to the cell membrane to facilitate the release of perforinand granzyme. Betts et al. (2003, J. Immunol. Methods 281:65-78),demonstrated that CD107 expression can be detected on an antigenspecific CD8 T cells as early as 30 minutes post stimulation withmaximal expression 4 hours post-stimulation. Thus, antigen specificeffectors can be identified without killing the desired T cells therebyidentifying which antigen is activating the cells. Similar studies toidentify and expand HIV specific T cells are performed as disclosedherein.

FIG. 4 illustrates, without wishing to be bound by any particulartheory, an experimental approach for expanding HIV specific CD8 T cellswith a broad specificity. Banked T cells and high titer, autologousviral isolates from a recently completed adoptive transfer clinicaltrial are available (Levine et al., 2002, Nature Med. 8:47-53) and areused in the methods disclosed herein. A patient's viral isolate isinactivated using 250 μM of aldrithiol-2 (AT-2) in order to preserve thefusogenic potential of the virus (Lu et al., 2001, J. Virol.75:8949-8956). The ability of AT-2 to inactivate HIV is validated byinfecting PHA blasts with this treated virus and measuring p24production over a two week period. In order to permit HIV fusion, thecell based aAPC that best allowed expansion of Pol-specific T cells istransduced with CD4 and CCR5 lentiviral expression vectors that arealready available (Simmons et al., 2003, Virology 305:115-123). K562cells naturally express CXCR4 (Gupta et al., 1999, J. Leukoc. Biol.66:135-143). These aAPCs are pulsed with the inactivated virus (50 ng ofp24/million cell based aAPC) for 2 hours at 37° C. (Lu et al., 2001, J.Virol. 75:8949-8956). Fifty million CD8 T cells from the patient aremixed with antigen loaded aAPCs in duplicate. With one culture, theutility of using CD107 mobilization as a surrogate of antigen specific Tcells endowed with enhanced effector functions is assessed. Four hoursafter stimulation, the cells are stained for CD8 and CD107, theCD8⁺CD107⁺ cells are sorted using the BLS 3 sorter and are expanded inisolation using “infected” aAPCs. With the other set of cells, theability of these aAPCs to selectively expand HIV specific T cells fromthe polyclonal population is tested. Chemically inactivated HIV infectedaAPCs can be used to stimulate the bulk CD8 T cells from the HIVinfected individual every 10 days. As a control, both aAPCs cells thathave not been pulsed with virus, which should only expand cells thatrecognize K562 antigens, and an αCD3-coated aAPC that expands all cells,are used. After two weeks of expansion, the cultures are monitored andthe cells are assessed to determine whether HIV-specific T cells thatrecognize the patient's own virus better than a reference strain of HIVare being enriched. PHA is used stimulate and superinfect the patient'sCD8 T cell depleted PBMCs with either high titer patient virus(approximately 5×10⁵ TCID₅₀/ml) or similarity high titer Bal (for R5patients) or NL4-3 (for X4 patients) viruses for 3 days. Most of thepatients that received ex vivo expanded, autologous CD4 T cells hadundetectable viral loads (Levine et al., 2002, Nature Med. 8:47-53) andthus after only 3 days the vast majority of replicating virus willrepresent the virus that was superinfected. Approximately 400,000 PBMCsobtained from an infected CD8 depleted patient are mixed with 100,000CD8 T cells expanded from aAPC infected with the patient's own virus.After 24 hours, the percentage of CD8 T cells expressing INF-λ andperforin is measured by intracellular flow cytometry. If more CD8 Tcells expressing IFN-λ and perforin is observed when mixed with thePBMCs superinfected with the patient's own virus compared with thosesuperinfected with Bal or NL4-3 reference strains, aAPC presentation ofthe patient virus likely allowed the expansion of T cells thatselectively recognize the patient's viral epitopes. This approach hastremendous potential to increase the breadth of a patient's HIV responseagainst his or her own virus and can potentially elicit responsesagainst cryptic epitopes not well presented during a natural HIVinfection (Sewell et al., 1999, J. Immunol. 162:7075-7079). Moreover,these experiments provide a determination of whether CD107 stainingprovides a more robust way to identify and expand HIV specific T cells.

It was previously demonstrated that high affinity CTLs were generatedfrom APCs expressing lower antigen levels (Alexander-Miller et al.,1996, Proc Natl. Acad. Sci U.S.A. 93:4102-4107; Oh et al., 2003, J.Immunol. 170:2523-2530). Whether aAPCs expressing lower levels of MHCclass I are more effective in generating high avidity T cells that havea greater potential to kill their targets is assessed. Based on thedisclosure herein, the amount of virus used to load the KA2 cells istitrated, or an aAPC expressing lower amounts of HLA-A2 is used, toensure that high-affinity T cell responses are generated that are usefulfor adoptive transfer immunotherapy (Alexander-Miller et al., 1996, ProcNatl. Acad. Sci. U.S.A. 93:4102-4107).

If the chemically inactivated virus loaded aAPC is not an effectivemethod to expand patient specific T cells, this method of generating HIVspecific T cells is compared with cross priming methods described byLarsson et al. (2002, AIDS 16:1319-1329). For this method to work, theK562 based aAPC possesses the ability to uptake antigens from dying ordead T cells. T cells are transduced with either GFP or flu matrix-GFPfusion LV construct. These cells are subjected to UVB radiation asdescribed in Schlienger et al. (2003, Clin. Cancer Res. 9:1517-1527),and mixed with the optimal HLA-A2 expressing aAPC. If the aAPC properlyprocesses the dead flu infected T cells, KA2 cells incubated with theapoptotic T cells expressing the flu matrix-GFP fusion protein, but notthe ones incubated with T cells expressing just GFP, can expand the fluspecific cells as described elsewhere herein. If the KA2 processes andpresents antigen from dead cells, a patient's T cells can besuperinfected with his/her virus. These cells are subjected to UVBradiation and are incubated with aAPCs. The ability of aAPCs loaded withapoptotic HIV infected cells to promote expansion of HIV specific Tcells is then evaluated as described elsewhere herein.

Alternatively, HIV-specific T cells with multiple specificities areexpanded using a panel of HIV-specific tetramers. In this approach, anarray of HIV-specific tetramers labeled with the same fluorochrome aremixed with T cells from an HIV infected donor and T cells that bindthese assorted tetramers are sorted into a single population. Thispopulation of antigen specific T cells is expanded using the optimalaAPC, as described elsewhere herein, and the ability of T cells expandedin this manner to recognize and respond to autologous virus versusreference strains are evaluated as described elsewhere herein.Currently, there are defined HLA-A2 tetramers that recognize conservedepitopes in gag, pol, and nef and new tetramers can be created thatpresent HIV-specific peptides. While this approach expands a limitednumber of antigen specific T cells, it should be sufficient to preventviral escape. Moreover, the methods disclosed herein can be rapidlytranslated into Phase I clinical trials.

Example 10 Production and Evaluation of K562 Cells Comprising CD32 orCD64

K562 cells were stably cotransfected with (i) the human Fcγ receptorCD32 to permit exogenous loading of anti-CD3 and anti-CD28 antibodies,and a separate population of cells was transduced with the human Fcγreceptor CD64, which permits high-affinity loading of anti-CD3,anti-CD28, and other receptors, and (ii) human 4-1BB ligand. 4-1BB, alsoknown as CD137, is a member of the TNF receptor family that promotessurvival of CD8⁺ T cells (Hurtado et al., 1997, J. Immunol.158:2600-2609; Takahashi et al., 1999, J. Immunol. 162:5037-5040; Tranet al., 1995, J. Immunol. 155:1000-1009). 4-1 BB stimulationpreferentially activates CD8 T cells in vitro, amplifies CTL responsesin vivo, and improves survival of activated CTLs (Shuford et al., 1997,J. Exp. Med. 186:47-55). 4-1BB is a candidate molecule that can promotelong-term ex vivo growth of CD8 T cells. The initial growth rate of CD8T cells stimulated with either CD3/28 beads or K32/4-1BBL aAPCs coatedwith anti-CD3 and CD28 Ab (K32/4-1BBL CD3/28) was equivalent. However,upon restimulation, only CD8 T cells activated with K32/4-1BBL CD3/28aAPCs continued to expand. This ability to expand correlated withupregulation of the cell survival gene Bcl-xL and the cytokine IL-2. Inthe absence of these genes being induced, a large percentage of thecultures became Annexin V positive, an early sign of apoptosis (Maus etal., 2002, Nature Biotechnol. 20:143-148). “Crosstalk” between the cellbased aAPCs and T cells was observed (Thomas et al., 2002, Clin.Immunol. 105:259-272).

Transduction of K562 cells with CD64 was accomplished as follows: K562cells were transduced with a lentiviral vector expressing CD64 (SEQ IDNO:2) according to the methods described herein. High expressors weresorted and single clones were screened for CD64 expression. One clonewas selected was further characterization (FIG. 14).

The binding capacity of K562 cells expressing CD64 (K64 cells) wasevaluated as follows. One million K64 cells were loaded with 0.5-50 μlof IgG2a-FITC-labeled antibodies for 1 hour at 4° C., then washed onceand fixed. Using the known antibody concentration (50 μg/ml), a knownFITC/protein ratio (3.5) and Immuno-Brite (Beckman Coulter) beads, theamount of antibody bound to the K64 cells was calculated to be betweenabout 2000 and 6000 antibodies bound to each cell (FIG. 15).

To evaluate the ability of K64 cells to load antibody and stimulate Tcells, K64 and K32 cells were irradiated at 100 Gy, and loaded with 1μg/ml of anti-CD3/anti-CD28 mixture per 10⁶ cells in the protein-freePFHM II media (Gibco/Invitrogen, Carlsbad, Calif.) and rotated at 4° C.for 1 hour. The cells were then washed three times with the same proteinfree media, resuspended in the same media, and added to CD4 T-cells at aratio of 2:1. T-cells were resuspended in RPMI+10% HSAB at aconcentration of 10⁶ cells per milliliter. As a control K64 and K32cells were also loaded using the conventional method (10 minutes at roomtemperature without washes), and then mixed with CD4 T-cells.

As illustrated in FIG. 16, when K64 cells are compared to K32 cells, K64cells loaded with anti-CD3/CD28 antibodies and washed three times toremove excess, unbound antibody are still capable of efficientlystimulating T-cells. Specifically, as depicted in FIG. 16, resting CD4 Tcells have mean cell volume of ˜140 fl. The disappearance of thispopulation of cells indicates that the CD4 T cells have becomeactivated. Thus, the aAPC cells of the present invention can be used inin vivo applications, as described elsewhere herein because they arecapable of initiating T cell stimulation and proliferation without thepresence of excess antibody that could result in a HAMA (humananti-mouse antibody) response if monoclonal antibodies are used in an invivo application of the present invention.

Moreover, as demonstrated in FIG. 17, much less antibody is required tooptimally load K64 cells, but washing K64 cells to prevent a HAMAreaction when administered to a mammal has minimal if any effect on theability of an aAPC to stimulate a T cell. K64 cells were irradiated at100 Gy, and loaded with either 1, ¼, 1/16, 1/64 or 1/256 mg/ml ofanti-CD3/antiCD28 mixture per 10⁶ cells in duplicate in protein-freePFHM II media (Gibco/Invitrogen, Carlsbad, Calif.) and rotated for 1hour at 4° C. One set of cells were washed three times with PFHM II andresuspended in the same media. The other set of cells were not washed.Both sets of K64 cells were added to CFSE labeled CD4 and CD8 T cells ata ratio 2:1. T-cells were resuspended in RPMI+10% HSAB, 10⁶/ml, asdescribed above. As a control K64 and K32 cells were also loaded usingconventional method (10 minutes at room temperature without washes), andthen mixed with CD4 T-cells. CFSE dilution was measured by flowcytometry.

As illustrated in FIG. 17, three washes to remove excess antibody haslittle effect of aAPC comprising CD64 to stimulate both CD4 and CD8cells.

Example 11 Expansion and Functional Characterization of Tregs

Naturally occurring CD25+CD4+ suppressor cells (Tregs) cells play anactive part in establishing and maintaining immunologicalunresponsiveness to self constituents (i.e., immunological selftolerance) and negative control of various immune responses to non-selfantigens. There are a paucity of reliable markers for defining Tregs,but naturally occurring CD25+CD4+ Tregs are the most widely studiedbecause accumulating evidence indicates that this population plays acrucial role in the maintenance of immunological self tolerance andnegative control of pathological as well as physiological immuneresponses. Their natural presence in the immune system as aphenotypically distinct population makes them a good target fordesigning ways to treat or prevent immunological diseases and to controlpathological as well as physiological immune responses. However, little,if any methods exist to expand and manipulate this population of cells.

In order to induce the stimulation and proliferation and investigate thefunctions of aAPC contacted Treg cells, the following experiments wereperformed.

Peripheral blood lymphocytes were labeled with anti-CD4 and anti-CD25antibody and the top 1% expressing CD25+ cells were isolated by cellsorting. These cells were stimulated with either anti-CD3 and CD28antibody coated beads or K32 cells loaded with anti-CD3 and CD28antibody. Cell expansion was measured by culturing the T cells in thepresence of 3000 U/ml of IL-2 and maintaining the T cell concentrationbetween 0.8 and 2 million cells per milliliter. Cells were counted onCoulter Counter IIE every two to three days. As illustrated in FIG. 18,aAPC stimulation of Treg populations resulted in a greater increase inthe number of cells when compared to bead stimulation. In addition,greater numbers of Treg cells are produced more quickly than withconventional means, such as beads.

To evaluate the functionality of Treg cells stimulated by aAPCs, CD4 andCD25 positive and CD4 CD25 negative cells were expanded for 17 daysusing K32 cells loaded with anti-CD3 and CD28 Ab and 3000 U/ml of IL-2.These cells were mixed with resting, CFSE stained cells from the samedonor at a 1:4 ratio (1 expanded cell for every 4 resting cells). Thismixture was placed on allogenic dendritic cells and CFSE dilution wasmeasured by flow cytometry. As illustrated in FIG. 19, Treg cellsexpanded with aAPCs suppress an allogeneic mixed lymphocyte reaction andthe expanded CD4-positive-CD25-positive suppressed T cell expansionwhereas the CD25 negative population did not.

A similar experiment was performed using aAPCs expressing CD32 (K32cells) expressing OX40L. As illustrated in FIG. 20, CD4+CD25+ Treg cellsare rendered non suppressive after such treatment with aAPCs.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method for specifically inducing proliferationof a T cell expressing a known co-stimulatory molecule, said methodcomprising contacting said T cell with an artificial presenting cell(aAPC) comprising at least one co-stimulatory ligand and CD64, whereinsaid CD64 is loaded with an anti-CD3 antibody, further wherein said CD64is expressed from an exogenous expression vector, thereby specificallyinducing proliferation of said T cell.
 2. A method for specificallyinducing proliferation of a T cell expressing a known co-stimulatorymolecule, said method comprising contacting a population of T cellscomprising at least one T cell expressing said known co-stimulatorymolecule with an aAPC comprising at least one co-stimulatory ligand andCD64, wherein said CD64 is loaded with an anti-CD3 antibody, furtherwherein said CD64 is expressed from an exogenous expression vector,wherein binding of said known co-stimulatory molecule with saidco-stimulatory ligand induces proliferation of said T cell.
 3. Themethod of claim 1, wherein said T cell is a CD8 T cell.
 4. The method ofclaim 2, wherein said T cell is a CD8 T cell.